A hybridoma-producing MAb reactive to G-Rb1 was obtained by general procedure and classified into IgG2b which had κ light chains [8]. The reactivity of IgG type MAb, 9G7 was tested by varying antibody concentration and by performing a dilution curve. The antibody concentration was selected for competitive ELISA. The free MAb following competition is bound to polystyrene microtiter plates precoated with G-Rb1-human serum albumin (HSA). Under these conditions, the full measurement range of the assay extends from 20 to 400 ng/mL. The cross-reactivity against G-Rc and G-Rd, which possess a diglucose moiety at‐ tached to the C-3 hydroxy group, were weak compared with G-Rb1 (0.024 and 0.020 %, re‐ spectively). G-Re and G-Rg1 showed no cross-reactivity (less than 0.005 %). It is evident that the MAb reacted only with a small number of structurally related G-Rb1 molecules, and very

*2.1.3. Preparation of anti-G-Rb1 immunoaffinity column and appropriate buffer systems for*

The purified IgG (10 mg) was treated by NaIO4 to give dialdehyde group in sugar moiety which was coupled to Affi-Gel Hz hydrazide gel resulting in a hydrozone-type immunoaf‐ finity gel [32]. The immunoaffinity gel was packed into plastic mini-column (Figure 2). Due to examine the optimal conditions of adsorption and elution, 400 μg of G-Rb1 was dissolved in phosphate buffered saline (PBS) and loaded on anti-G-Rb1 affinity column. After washing with washing buffer (20 mM PB containing 0.5 M NaCl), various buffer solutions for elution were loaded on the column, and then the recovery efficiency was determined by ELISA. The G-Rb1 concentration was somewhat increased by eluting with a 20 mM phosphate buffer containing 0.5 M KSCN and 10 % MeOH. When the 20 mM phosphate buffer was changed to 100 mM AcOH buffer (pH 4), the elution ability reached the optimal level. Although 20 % MeOH could enhance the elution of G-Rb1, higher MeOH concentration of over 20 % was ineffective. Thus, 100 mM AcOH buffer containing 0.5 M KSCN and 20 % MeOH could be

#### *2.2.1. One-step purification of G-Rb1 from crude extract of P. ginseng roots by anti-G-Rb1 immunoaffinity column*

A crude extract (3.8 mg) of *P. ginseng* roots was loaded onto anti-G-Rb1 immunoaffinity col‐ umn. The column was washed with the washing buffer (fractions 1-20), and then eluted with elution buffer (fractions 21-40). As shown in Figure 3, the fractions 1-8 contained the overloaded G-Rb1, which determined by ELISA. The other ginsenosides such as G-Rg1, Rc, Re and Rd were also detected in these fractions by Eastern blotting procedure. After wash‐ ing, a sharp peak was observed around fractions 21-24 of elution buffer, which contained G-Rb1. However, these eluted fractions were still contaminated by a small amount of malonyl-G-Rb1 as detected by Eastern blotting. The malonyl-G-Rb1 has almost the same crossreactivity with G-Rb1 [32]. Therefore, the eluted fractions were treated with a mild alkaline solution (0.1 % KOH in MeOH) at room temperature to give pure G-Rb1 [19]. Overcharged G-Rb1 in washing solution (fractions 1-8) was repeatedly loaded and finally isolated in pure form. The anti-G-Rb1 MAb was stable during all procedures, and the immunoaffinity col‐ umn showed almost no decrease in capacity (20 μg of G-Rb1/ml gel) after repeated use more than 10 times under same conditions, as reported for a one-step purification of forskolin from a crude extract of *Coleus forslohlii* root [17].

**Figure 3.** Elution profile of *P. ginseng* crude extract separated by anti-G-Rb1 immunoaffinity column. The concentra‐ tion of G-Rb1 in each fraction was monitoring by ELISA using anti-G-Rb1 MAb. Individual fraction (2 mL) were assayed by ELISA. a Inhibition = (A0-A)/A0 ;A0 is the absorbance in the absence of the test compounds. A is the absence in the presence the test compounds.

This methodology is effective for the rapid and simple purification of G-Rb1 and may open up a wide field of comparable studies with other families of saponins for which an accepta‐ ble method for one-step separation has not yet been developed. Furthermore, to separate the total ginseng saponins, a wide cross-reactive MAb against ginsenoside, like anti G-Re MAb which showed wide cross-reactivity, could be designed [34]. A combination of immunoaf‐ finity column, Eastern blotting and ELISA could be used to survey low concentrations of ginsenoside Rb1 of plant origin and/or in experimental animals and human. In fact we have succeeded in the isolation of G-Rb1 from a different plant, *Kalopanax pictus* Nakai, which was not known previously to contain ginsenosides, using this combination of methods [35].

ed fraction by the washing buffer, and showed many spots, indicating chikusetsusaponins, similar to the original extract of *P. japonicus*. After washing, the column was eluted by elu‐ tion buffer (fraction 5), and then one spot was detected. As shown in Figure 4B, Eastern blot‐ ting indicated two different spots in washing fraction (Compound 1) and eluted fraction (Compound 2). These compounds bound with anti-G-Rb1 MAb have a dammarane saponin

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35

**Figure 4.** Purification and determination of ginsenosides of *P. japonicus* by the anti-G-Rb1 immunoaffinity column. TLC (A) and Eastern blotting (B) profiles of the separated fractions from *P. japonicus* crude extract by the anti-G-Rb1 immu‐ noaffinity column. Lane S indicates the standard of ginsenosides (G-Rd, G-Rc, G-Rb1, G-Rg1, and G-Re). Lane 1-4 and

**Figure 5.** Chemical structures of G-Rb1 and chikusetsusaponins purified from *P. japonicus* by the anti-G-Rb1 immunoaf‐

Compound 1 has three sugar moieties in a molecule because that the *R* F value closes to that of G-Rd, indicating that this compound is chikusetsusaponin III (Figure 5). Finally, we iden‐ tified that this compound as chikusetsusaponin III in a direct comparison with authentic sample [32]. Another unknown spot, compound 2 appeared in fraction 5 of the eluted frac‐ tion. Thus, compound 2 has a similar molecular structure and high cross-reactivity with G-Rb1, and seems to be related ginseng saponin having protopanaxadiol as an aglycone. Moreover, compound 2 might have the same sugar fragments and possess five sugar moiety compared with G-Rb1, as indicated by their *R* F value. From these evidences compound 2

having protopanaxadiol as a framework.

Lane 5 were the washing fractions and the eluted fraction, respectively.

finity column.

#### *2.2.2. Isolation and determination of unknown compounds related to G-Rb1 by anti-G-Rb1 immunoaffinity column*

Several species of ginseng are known to exist and contain different amount and kinds of gin‐ senosides. *P. japonicus* is distributed in Japan and China and it is morphologically different from the other *Panax* species. Yahara *et al.* indicated that G-Rbl was not detected in *P. japoni‐ cus*, and isolated oleanane-type saponins called chikusetsusaponins and determined their structures [36]. Morita *et al*. reported the varieties of saponins in *P. japonicus* by chemical analysis. These results suggested that the concentration of G-Rb1 might be trace level in *P. japonicas* [37]. We previously analyzed the G-Rb1 concentration in several ginseng roots by ELISA using anti-G-Rb1 MAb and HPLC after pre-treatment under mildly alkaline condition [32]. As shown in Table 1, G-Rb1 concentrationsof *P. ginseng*, *P. notoginseng* and *P. quinquefo‐ lius* were correlated between ELISA and HPLC. However, the G-Rb1 of *P. japonicus* was higher concentrations compared with HPLC and previous reports [37]. This data suggest that anti-G-Rb1 MAb using ELISA has the cross-reactivity with some unknown compounds contained in *P. japonicus*.

To clarify the unknown compounds bound to anti-G-Rb1 MAb, the crude extract of *P. japoni‐ cus* was concentrated by immunoaffinity column using anti-G-Rb1 MAb. The crude root ex‐ tract was loaded on the column and washed with the washing Buffer, followed by the elution buffer as already indicated. Figure 4 shows the H2SO4 staining (A) and the Eastern blotting (B) profiles of the washing fractions 1-4 and eluted fraction 5. Fraction 1 is first elut‐ ed fraction by the washing buffer, and showed many spots, indicating chikusetsusaponins, similar to the original extract of *P. japonicus*. After washing, the column was eluted by elu‐ tion buffer (fraction 5), and then one spot was detected. As shown in Figure 4B, Eastern blot‐ ting indicated two different spots in washing fraction (Compound 1) and eluted fraction (Compound 2). These compounds bound with anti-G-Rb1 MAb have a dammarane saponin having protopanaxadiol as a framework.

total ginseng saponins, a wide cross-reactive MAb against ginsenoside, like anti G-Re MAb which showed wide cross-reactivity, could be designed [34]. A combination of immunoaf‐ finity column, Eastern blotting and ELISA could be used to survey low concentrations of ginsenoside Rb1 of plant origin and/or in experimental animals and human. In fact we have succeeded in the isolation of G-Rb1 from a different plant, *Kalopanax pictus* Nakai, which was not known previously to contain ginsenosides, using this combination of methods [35].

Several species of ginseng are known to exist and contain different amount and kinds of gin‐ senosides. *P. japonicus* is distributed in Japan and China and it is morphologically different from the other *Panax* species. Yahara *et al.* indicated that G-Rbl was not detected in *P. japoni‐ cus*, and isolated oleanane-type saponins called chikusetsusaponins and determined their structures [36]. Morita *et al*. reported the varieties of saponins in *P. japonicus* by chemical analysis. These results suggested that the concentration of G-Rb1 might be trace level in *P. japonicas* [37]. We previously analyzed the G-Rb1 concentration in several ginseng roots by ELISA using anti-G-Rb1 MAb and HPLC after pre-treatment under mildly alkaline condition [32]. As shown in Table 1, G-Rb1 concentrationsof *P. ginseng*, *P. notoginseng* and *P. quinquefo‐ lius* were correlated between ELISA and HPLC. However, the G-Rb1 of *P. japonicus* was higher concentrations compared with HPLC and previous reports [37]. This data suggest that anti-G-Rb1 MAb using ELISA has the cross-reactivity with some unknown compounds

> **Sample ELISA HPLC** *P. ginseng* **White ginseng 5.49 ±0.75 4.96 ± 0.05**

> *P. notoginseng* **47.08 ± 3.34 42.39 ± 1.39** *P. quinquefolium* **48.51 ± 1.79 47.96 ± 1.04** *P. japonicus* **1.37 ± 0.34 0.63 ± 0.06**

To clarify the unknown compounds bound to anti-G-Rb1 MAb, the crude extract of *P. japoni‐ cus* was concentrated by immunoaffinity column using anti-G-Rb1 MAb. The crude root ex‐ tract was loaded on the column and washed with the washing Buffer, followed by the elution buffer as already indicated. Figure 4 shows the H2SO4 staining (A) and the Eastern blotting (B) profiles of the washing fractions 1-4 and eluted fraction 5. Fraction 1 is first elut‐

**Red ginseng 3.57 ± 0.62 3.93 ± 0.34 Fibrous ginseng 64.44 ± 3.64 69.75± 1.45**

**G-Rb1 concentration (µg/mg dry weight powder)**

*2.2.2. Isolation and determination of unknown compounds related to G-Rb1 by anti-G-Rb1*

*immunoaffinity column*

34 Latest Research into Quality Control

contained in *P. japonicus*.

**Table 1.** G-Rb1 concentration in various ginseng samples

**Figure 4.** Purification and determination of ginsenosides of *P. japonicus* by the anti-G-Rb1 immunoaffinity column. TLC (A) and Eastern blotting (B) profiles of the separated fractions from *P. japonicus* crude extract by the anti-G-Rb1 immu‐ noaffinity column. Lane S indicates the standard of ginsenosides (G-Rd, G-Rc, G-Rb1, G-Rg1, and G-Re). Lane 1-4 and Lane 5 were the washing fractions and the eluted fraction, respectively.

**Figure 5.** Chemical structures of G-Rb1 and chikusetsusaponins purified from *P. japonicus* by the anti-G-Rb1 immunoaf‐ finity column.

Compound 1 has three sugar moieties in a molecule because that the *R* F value closes to that of G-Rd, indicating that this compound is chikusetsusaponin III (Figure 5). Finally, we iden‐ tified that this compound as chikusetsusaponin III in a direct comparison with authentic sample [32]. Another unknown spot, compound 2 appeared in fraction 5 of the eluted frac‐ tion. Thus, compound 2 has a similar molecular structure and high cross-reactivity with G-Rb1, and seems to be related ginseng saponin having protopanaxadiol as an aglycone. Moreover, compound 2 might have the same sugar fragments and possess five sugar moiety compared with G-Rb1, as indicated by their *R* F value. From these evidences compound 2 might be chikusetsusaponin III-20-*O*-gentiobiose, chikusetsusaponin VI (Figure 5), which has 5 sugars in a molecule in good agreement with the *R* F value previously reported [38] and we confirmed that compound 2 is chikusetsusaponin VI by the direct comparison with authentic sample.

**3. Glycyrrhizin-knockout extract and its application for** *in vitro* **assay**

application for functional analysis of GC in licorice crude extract.

tive ELISA and Eastern blotting method using anti-GC MAb [11,49].

unbound fraction "GC-knockout (GC-KO) extract" [50].

**characterization of GC-KO extract**

**3.1. Preparation of GC-KO extract by anti-GC immunoaffinity column and the**

Our previous study demonstrated the preparation of anti-GC MAb [11]. The cross-reactivi‐ ties of the anti-GC MAb against glycyrrhetic acid-3-*O*-glucuronide and glycyrrhetic acid were 0.585 % and 1.865 %, respectively. The other related compounds (deoxycholic acid, ur‐ solic acid, and oleanolic acid) were all less than 0.005 %. Moreover, we established competi‐

The immunoaffinity column against GC was prepared by coupling the purified 60 mg of the anti-GC MAb to 25 ml of an Affi-Gel Hz gel [11]. To eliminate GC from licorice extract, 12 mg of licorice crude extract (GC content: 1275.0 μg) in loading buffer (5 % MeOH) was ap‐ plied on the anti-GC MAb immunoaffinity column, and then the loading buffer was contin‐ uously circulated through the column to enhance the binding efficiency. After overnight circulation at 4 °C, the unbound fraction was separated. The column was washed with washing buffer (5 % MeOH) and then eluted with elution buffer (20 mM phosphate buffer containing 30 % MeOH). After separation, each fraction was deionized and the solvent was lyophilized. Figure 7 showed the recovery ratio of GC checked by ELISA. In the unbound fraction, 3.50 μg of GC (0.27% of the applied GC) was detected. On the other hand, 1269.26 μg of GC (99.55% of the applied GC) was obtained in the bound fraction. These data indicate that the anti-GC column could eliminate 99.55 % of the loading GC. Thus, we named this

Licorice (*Glycyrrhiza* spp.) is also important crude drug used in over 70 % of the TCMs and Japanese Kampo medicines. It is prescribed with other herbal medicines as a demulcent in the treatment of sore throats, an expectorant for coughs and bronchial catarrh, an antitus‐ sive, a taste-modifying agent for relieving pain, an anti-inflammatory agent for anti-allergic reactions, rheumatism and arthritis, a prophylactic for liver disease and tuberculosis and adrenocorticoid insufficiency [41-43]. Accumulated evidence indicated that GC, a main sap‐ onin component of licorice, is one of the biologically active compounds. It has been reported that GC exhibits numerous pharmacological effects such as anti-inflammation, anti-ulcer, anti-tumor, anti-allergy, and hepatoprotective activities [44,45]. Clinically, GC has been used to treat patients with chronic hepatitis [46,47]. Although GC is supposed to be a major active principle in licorice crude extract, a number of studies by HPLC profiles suggested that lico‐ rice has many other bioactive components, including flavonoids, isoflavonoids and chal‐ cones [43,48]. Biological studies showed that various flavonoid glycosides and their aglycones of licorice exhibit anti-inflammatory, anti-oxidative, anti-microbial, superoxide scavenging, and anti-carcinogenic activities [43,48]. In order to confirm the role of GC in TCM, we previously purified GC from TCM using an immunoaffinity column conjugated with anti-GC MAb [20]. In this section, we describe the preparation of GC-KO extract and its

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These data suggested that the anti-G-Rb1 immunoaffinity column could isolate some un‐ known structurally resemble compounds having cross-reactivity against anti-G-Rb1MAb. Therefore, this purification system will be applied to survey new compounds related to target compound of MAb. In our previous studies, we demonstrated the immunoaffinity purification against all solasodine glycosides from crude extract by one-step purification. In this case, all solasodine glycoside have almost same cross-reactivity against anti-solamar‐ gine MAb [12].

#### *2.2.3. Preparation of G-Rb1 knockout extract by anti-G-Rb1 immunoaffinity column*

The capacity of this anti-G-Rb1immunoaffinity column is 20 μg of G-Rb1/ml gel [32]. By load‐ ing the samples not to exceed the binding capacity against G-Rb1, this immunoaffinity col‐ umn becomes possible to remove all G-Rb1 from crude ginseng extract. Figure 6 showed H2SO4 staining of TLC of the purification steps by the immunoaffinity column. Lane 1 and 2 were spotted the standard of ginsenosides (G-Rd, G-Rc, G-Rb1, G-Rg1, and G-Re). Lane A, B, and C were the crude extract, the washing fraction, and the eluted fraction, respectively. In the crude extract (lane A), all spots of ginsenosides were clearly detected. On the other hand, the washing fraction (lane B) contained all of the ginsenosides in the crude extract except G-Rb1. Furthermore, the spot of G-Rb1 was detected in the eluted fractions (lane C). These data strongly indicated that G-Rb1 molecule in the ginseng extract can be eliminated by an anti-G-Rb1 immunoaffinity column and the washing fractions was knockout only by the antigen molecule, G-Rb1. Thus, we named the washing fractions a knockout (KO) extract [39,40]. This KO extract may be useful for the determination of real pharmacologically active princi‐ ple in the TCMs.

**Figure 6.** Preparation of G-Rb1-KO extract from *P. ginseng* crude extract using anti-G-Rb1 immunoaffinity column. Lane S1 and S2 indicate the standard of ginsenosides (G-Rd, G-Rc, G-Rb1, G-Rg1 and G-Re). Lane A, B, and C were the crude extract, the washing fraction, and the eluted fraction, respectively.

### **3. Glycyrrhizin-knockout extract and its application for** *in vitro* **assay**

might be chikusetsusaponin III-20-*O*-gentiobiose, chikusetsusaponin VI (Figure 5), which has 5 sugars in a molecule in good agreement with the *R* F value previously reported [38] and we confirmed that compound 2 is chikusetsusaponin VI by the direct comparison with

These data suggested that the anti-G-Rb1 immunoaffinity column could isolate some un‐ known structurally resemble compounds having cross-reactivity against anti-G-Rb1MAb. Therefore, this purification system will be applied to survey new compounds related to target compound of MAb. In our previous studies, we demonstrated the immunoaffinity purification against all solasodine glycosides from crude extract by one-step purification. In this case, all solasodine glycoside have almost same cross-reactivity against anti-solamar‐

The capacity of this anti-G-Rb1immunoaffinity column is 20 μg of G-Rb1/ml gel [32]. By load‐ ing the samples not to exceed the binding capacity against G-Rb1, this immunoaffinity col‐ umn becomes possible to remove all G-Rb1 from crude ginseng extract. Figure 6 showed H2SO4 staining of TLC of the purification steps by the immunoaffinity column. Lane 1 and 2 were spotted the standard of ginsenosides (G-Rd, G-Rc, G-Rb1, G-Rg1, and G-Re). Lane A, B, and C were the crude extract, the washing fraction, and the eluted fraction, respectively. In the crude extract (lane A), all spots of ginsenosides were clearly detected. On the other hand, the washing fraction (lane B) contained all of the ginsenosides in the crude extract except G-Rb1. Furthermore, the spot of G-Rb1 was detected in the eluted fractions (lane C). These data strongly indicated that G-Rb1 molecule in the ginseng extract can be eliminated by an anti-G-Rb1 immunoaffinity column and the washing fractions was knockout only by the antigen molecule, G-Rb1. Thus, we named the washing fractions a knockout (KO) extract [39,40]. This KO extract may be useful for the determination of real pharmacologically active princi‐

**Figure 6.** Preparation of G-Rb1-KO extract from *P. ginseng* crude extract using anti-G-Rb1 immunoaffinity column. Lane S1 and S2 indicate the standard of ginsenosides (G-Rd, G-Rc, G-Rb1, G-Rg1 and G-Re). Lane A, B, and C were the

crude extract, the washing fraction, and the eluted fraction, respectively.

*2.2.3. Preparation of G-Rb1 knockout extract by anti-G-Rb1 immunoaffinity column*

authentic sample.

36 Latest Research into Quality Control

gine MAb [12].

ple in the TCMs.

Licorice (*Glycyrrhiza* spp.) is also important crude drug used in over 70 % of the TCMs and Japanese Kampo medicines. It is prescribed with other herbal medicines as a demulcent in the treatment of sore throats, an expectorant for coughs and bronchial catarrh, an antitus‐ sive, a taste-modifying agent for relieving pain, an anti-inflammatory agent for anti-allergic reactions, rheumatism and arthritis, a prophylactic for liver disease and tuberculosis and adrenocorticoid insufficiency [41-43]. Accumulated evidence indicated that GC, a main sap‐ onin component of licorice, is one of the biologically active compounds. It has been reported that GC exhibits numerous pharmacological effects such as anti-inflammation, anti-ulcer, anti-tumor, anti-allergy, and hepatoprotective activities [44,45]. Clinically, GC has been used to treat patients with chronic hepatitis [46,47]. Although GC is supposed to be a major active principle in licorice crude extract, a number of studies by HPLC profiles suggested that lico‐ rice has many other bioactive components, including flavonoids, isoflavonoids and chal‐ cones [43,48]. Biological studies showed that various flavonoid glycosides and their aglycones of licorice exhibit anti-inflammatory, anti-oxidative, anti-microbial, superoxide scavenging, and anti-carcinogenic activities [43,48]. In order to confirm the role of GC in TCM, we previously purified GC from TCM using an immunoaffinity column conjugated with anti-GC MAb [20]. In this section, we describe the preparation of GC-KO extract and its application for functional analysis of GC in licorice crude extract.

#### **3.1. Preparation of GC-KO extract by anti-GC immunoaffinity column and the characterization of GC-KO extract**

Our previous study demonstrated the preparation of anti-GC MAb [11]. The cross-reactivi‐ ties of the anti-GC MAb against glycyrrhetic acid-3-*O*-glucuronide and glycyrrhetic acid were 0.585 % and 1.865 %, respectively. The other related compounds (deoxycholic acid, ur‐ solic acid, and oleanolic acid) were all less than 0.005 %. Moreover, we established competi‐ tive ELISA and Eastern blotting method using anti-GC MAb [11,49].

The immunoaffinity column against GC was prepared by coupling the purified 60 mg of the anti-GC MAb to 25 ml of an Affi-Gel Hz gel [11]. To eliminate GC from licorice extract, 12 mg of licorice crude extract (GC content: 1275.0 μg) in loading buffer (5 % MeOH) was ap‐ plied on the anti-GC MAb immunoaffinity column, and then the loading buffer was contin‐ uously circulated through the column to enhance the binding efficiency. After overnight circulation at 4 °C, the unbound fraction was separated. The column was washed with washing buffer (5 % MeOH) and then eluted with elution buffer (20 mM phosphate buffer containing 30 % MeOH). After separation, each fraction was deionized and the solvent was lyophilized. Figure 7 showed the recovery ratio of GC checked by ELISA. In the unbound fraction, 3.50 μg of GC (0.27% of the applied GC) was detected. On the other hand, 1269.26 μg of GC (99.55% of the applied GC) was obtained in the bound fraction. These data indicate that the anti-GC column could eliminate 99.55 % of the loading GC. Thus, we named this unbound fraction "GC-knockout (GC-KO) extract" [50].

**3.2.** *in vitro* **Assay by GC-KO extract prepared by anti-GC immunoaffinity column**

expression may be useful strategy to treat a variety of inflammatory diseases.

ly suppressed [50].

Nitric oxide (NO), synthesized by NO synthase (NOS) from L-arginine, is an important reg‐ ulatory/modulatory mediator for several physiological processes [51]. However, during in‐ flammatory process, a large amount of NO is produced by inducible NOS (iNOS) stimulated by bacterial lipopolysaccharide (LPS) and inflammatory cytokines participates in the patho‐ genesis of inflammatory diseases [52]. Overproduced NO synthesized by iNOS triggers a variety of inflammatory diseases including sepsis, psoriasis, arthritis, multiple sclerosis, and systemic lupus *erythematosus* [53]*.* Therefore, inhibiting NO production by blocking iNOS

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In LPS-treated mouse RAW264 macrophages, licorice extract inhibited NO production and iNOS expression. At 100 μg/mL of licorice extract, iNOS protein and mRNA were complete‐

**Figure 9.** Effect of GC-KO extract and the combination of GC-KO extract and GC on NO production (A) and iNOS pro‐ tein expression (B) in LPS-treated RAW264 cells.Each bar indicates the mean S.D. of four individual experiments.

We next examined the inhibitory effect of GC alone, GC-KO extract and the combined treat‐ ment with GC and GC-KO extract on NO production [50]. Since 100 μg of licorice extract contains 10.6 ±0.618 μg of GC,the cells were pre-treated with licorice extract (100 μg/ml), GC-KO extract (89.4 μg/ml), or the combination of GC-KO extract (89.4 μg/ml) and GC (10.6 μg/ml). Figure9A indicated that the treatment of licorice extract led to a marked suppression of NO production as compared to LPS treatment [inhibition ratio (IR) 57.7% ]. Interestingly, the inhibitory effect of GC-KO extract was lower (IR 17.8% ) compared with licorice extract although GC alone could not block NO production as indicated above. On the other hand, the combined treatment with GC-KO extract and GC significantly improved the inhibitory ability (IR 33.5% ). To determine whether the combinational effect of GC-KO extract and GC was related to iNOS expression, we performed Western blotting. As shown in Figure 9B, the treatment of GC-KO extract diminished the inhibitory ability of LE on iNOS expression, and addition of GC to GC-KO extract could improve it. These data suggest that GC alone cannot

\**P*<0.05, \*\**P*<0.01, \*\*\**P*<0.001 indicate significant differences from the LPS alone

**Figure 7.** Preparation of GC-KO extract from licorice extract by anti-GC immunoaffinity column.

To further characterize GC-KO extract, the TLC analysis and Eastern blotting were per‐ formed [50]. As shown in Figure 8A, several spots including GC were detected in lico‐ rice extract (Lane B). However, the spot of GC was completely disappeared in GC-KO extract, although all other spots were clearly detected (lane C). Eastern blotting by anti-GC MAb indicated that GC was detected in licorice extract (Figure 8B, lane B), but the spot of GC was disappeared in GC-KO extract (Figure 8B, lane C). Therefore, these data suggest that GC was specifically eliminated from licorice extract by anti-GC MAb immu‐ noaffinity column.

**Figure 8.** TLC profiles (A) and Eastern blotting by anti-GC MAb (B). Lane A, B, and C indicate GC, licorice extract, and GC-KO extract, respectively.

#### **3.2.** *in vitro* **Assay by GC-KO extract prepared by anti-GC immunoaffinity column**

Nitric oxide (NO), synthesized by NO synthase (NOS) from L-arginine, is an important reg‐ ulatory/modulatory mediator for several physiological processes [51]. However, during in‐ flammatory process, a large amount of NO is produced by inducible NOS (iNOS) stimulated by bacterial lipopolysaccharide (LPS) and inflammatory cytokines participates in the patho‐ genesis of inflammatory diseases [52]. Overproduced NO synthesized by iNOS triggers a variety of inflammatory diseases including sepsis, psoriasis, arthritis, multiple sclerosis, and systemic lupus *erythematosus* [53]*.* Therefore, inhibiting NO production by blocking iNOS expression may be useful strategy to treat a variety of inflammatory diseases.

In LPS-treated mouse RAW264 macrophages, licorice extract inhibited NO production and iNOS expression. At 100 μg/mL of licorice extract, iNOS protein and mRNA were complete‐ ly suppressed [50].

**Figure 7.** Preparation of GC-KO extract from licorice extract by anti-GC immunoaffinity column.

noaffinity column.

38 Latest Research into Quality Control

GC-KO extract, respectively.

To further characterize GC-KO extract, the TLC analysis and Eastern blotting were per‐ formed [50]. As shown in Figure 8A, several spots including GC were detected in lico‐ rice extract (Lane B). However, the spot of GC was completely disappeared in GC-KO extract, although all other spots were clearly detected (lane C). Eastern blotting by anti-GC MAb indicated that GC was detected in licorice extract (Figure 8B, lane B), but the spot of GC was disappeared in GC-KO extract (Figure 8B, lane C). Therefore, these data suggest that GC was specifically eliminated from licorice extract by anti-GC MAb immu‐

**Figure 8.** TLC profiles (A) and Eastern blotting by anti-GC MAb (B). Lane A, B, and C indicate GC, licorice extract, and

**Figure 9.** Effect of GC-KO extract and the combination of GC-KO extract and GC on NO production (A) and iNOS pro‐ tein expression (B) in LPS-treated RAW264 cells.Each bar indicates the mean S.D. of four individual experiments. \**P*<0.05, \*\**P*<0.01, \*\*\**P*<0.001 indicate significant differences from the LPS alone

We next examined the inhibitory effect of GC alone, GC-KO extract and the combined treat‐ ment with GC and GC-KO extract on NO production [50]. Since 100 μg of licorice extract contains 10.6 ±0.618 μg of GC,the cells were pre-treated with licorice extract (100 μg/ml), GC-KO extract (89.4 μg/ml), or the combination of GC-KO extract (89.4 μg/ml) and GC (10.6 μg/ml). Figure9A indicated that the treatment of licorice extract led to a marked suppression of NO production as compared to LPS treatment [inhibition ratio (IR) 57.7% ]. Interestingly, the inhibitory effect of GC-KO extract was lower (IR 17.8% ) compared with licorice extract although GC alone could not block NO production as indicated above. On the other hand, the combined treatment with GC-KO extract and GC significantly improved the inhibitory ability (IR 33.5% ). To determine whether the combinational effect of GC-KO extract and GC was related to iNOS expression, we performed Western blotting. As shown in Figure 9B, the treatment of GC-KO extract diminished the inhibitory ability of LE on iNOS expression, and addition of GC to GC-KO extract could improve it. These data suggest that GC alone cannot suppress iNOS expression, but combinational inhibition of iNOS expression may occur when GC coexists with the other constituents contained in licorice extract. The *in vitro* and in *vivo* analysis by using KO extract prepared by immunoaffinity column is a useful approach for determination of potential function of natural compound on *in vitro* and in *vivo* assays.

**References**

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[2] Tan, T. W., Huo, Q., & Ling, Q. (2002). Purification of glycyrrhizin from glycyrrhiza uralensis fisch with ethanol/phosphate aqueous two phase system. *Biochem Lett*,

[3] Sakata, R., Shoyama, Y., & Murakami, H. (1994). Production of monoclonal antibod‐ ies and enzyme immunoassay for typical adenylate cyclase activator, Forskolin. *Cyto‐*

[4] Xuan, L., Tanaka, H., Xu, Y., & Shoyama, Y. (1999). Preparation of monoclonal anti‐

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[6] Shoyama, Y., Fukada, T., & Murakami, H. (1996). Production of monoclonal antibod‐

[7] Kim, J. S., Tanaka, H., & Shoyama, Y. (2004). Immunoquantitative analysis for ber‐ berine and its related compounds using monoclonal antibodies in herbal medicines.

[8] Tanaka, H., Fukuda, N., & Shoyama, Y. (1999). Formation of monoclonal antibody against a major ginseng component, ginsenoside Rb1 and its characterization. Cyto‐

[9] Fukuda, N., Tanaka, H., & Shoyama, Y. (2000). Formation of monoclonal antibody against a major ginseng component, ginsenoside Rg1 and its characterization Mono‐

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clonal antibody for a ginseng saponin. *Cytotechnology*, 34(3), 197-204.

#### **4. Conclusion**

In this chapter, we introduce the unique strategy of one-step purification of target com‐ pounds from crude extract by anti-natural compound specific MAb-conjugated immunoaf‐ finity column. The immunoaffinity column conjugated with anti-G-Rb1 MAb could purify the G-Rb1 from *P. ginseng* extract, and the washing fraction contained all compounds ex‐ pect only G-Rb1, which was named G-Rb1-KO extract. By the use of the cross-reactivity of MAb, the anti-G-Rb1 immunoaffinity column can identify new unknown compounds relat‐ ed to target compound of MAb and determine their structures. Furthermore, our data suggest that the combination of the immunoaffinity column and ELISA by using MAb provided a reliable and high sensitivity analysis for target compound in various TCMs and crude extract. We also demonstrated the in vitro assay by using GC-KO extract prepared by anti-GC immunoaffinity column from licorice extract. The KO extract may be able to support the pharmacological investigation for finding out a really active compound in a TCM and crude drug.

#### **Acknowledgements**

This work was funded by the Asahi Beer Science Promoting Foundation and Takeda Science Foundation. The research in this paper was also supported in part by Sasakawa Scientific Research Grant from Japan Science Society and "Science and Technology Research Partner‐ ship for Sustainable Development (SATREPS)" supported by the Japan Science and Technol‐ ogy Agency (JST) and the Japan International Cooperation Agency (JICA).

#### **Author details**

Takuhiro Uto1 , Nguyen Huu Tung1 , Hiroyuki Tanaka2 and Yukihiro Shoyama1\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Faculty of Pharmaceutical Sciences, Nagasaki International University,, Japan

2 Faculty of Pharmaceutical Sciences, Kyushu University,, Japan

#### **References**

suppress iNOS expression, but combinational inhibition of iNOS expression may occur when GC coexists with the other constituents contained in licorice extract. The *in vitro* and in *vivo* analysis by using KO extract prepared by immunoaffinity column is a useful approach for determination of potential function of natural compound on *in vitro* and in *vivo* assays.

In this chapter, we introduce the unique strategy of one-step purification of target com‐ pounds from crude extract by anti-natural compound specific MAb-conjugated immunoaf‐ finity column. The immunoaffinity column conjugated with anti-G-Rb1 MAb could purify the G-Rb1 from *P. ginseng* extract, and the washing fraction contained all compounds ex‐ pect only G-Rb1, which was named G-Rb1-KO extract. By the use of the cross-reactivity of MAb, the anti-G-Rb1 immunoaffinity column can identify new unknown compounds relat‐ ed to target compound of MAb and determine their structures. Furthermore, our data suggest that the combination of the immunoaffinity column and ELISA by using MAb provided a reliable and high sensitivity analysis for target compound in various TCMs and crude extract. We also demonstrated the in vitro assay by using GC-KO extract prepared by anti-GC immunoaffinity column from licorice extract. The KO extract may be able to support the pharmacological investigation for finding out a really active compound in a

This work was funded by the Asahi Beer Science Promoting Foundation and Takeda Science Foundation. The research in this paper was also supported in part by Sasakawa Scientific Research Grant from Japan Science Society and "Science and Technology Research Partner‐ ship for Sustainable Development (SATREPS)" supported by the Japan Science and Technol‐

, Hiroyuki Tanaka2

1 Faculty of Pharmaceutical Sciences, Nagasaki International University,, Japan

and Yukihiro Shoyama1\*

ogy Agency (JST) and the Japan International Cooperation Agency (JICA).

, Nguyen Huu Tung1

\*Address all correspondence to<EMAIL_ADDRESS>
2 Faculty of Pharmaceutical Sciences, Kyushu University,, Japan

**4. Conclusion**

40 Latest Research into Quality Control

TCM and crude drug.

**Acknowledgements**

**Author details**

Takuhiro Uto1

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**Chapter 3**

**Standard Operating Procedures (SOP) for the**

**Compounds Contained in Plant Samples**

Elba Lúcia Cavalcanti de Amorim,

Joabe Gomes de Melo,

http://dx.doi.org/10.5772/51686

standardisation of the final product.

ologies for such determinations.

**1. Introduction**

Valérium Thijan Nobre de Almeida de Castro,

Allan Jonathan Chernichiarro Corrêa and

Additional information is available at the end of the chapter

Tadeu José da Silva Peixoto Sobrinho

**Spectrophotometric Determination of Phenolic**

The quality control of raw materials and products from plants is one of the topics most discussed by universities and health surveillance agencies. One of the primary tools used to ensure the reliability of production processes is the use of Standard Operating Procedures (SOPs). SOPs sequentially describe the steps of a particular methodology so that it can be reproduced by different analysts, which minimises variations in their implementation and improves the

Several techniques, such as high performance liquid chromatography, gas chromatography and mass spectrometry, can be used in SOPs to control the quality of plant phenolic com‐ pounds [1,2]. However, these compounds have a characteristic spectrum produced by the double bonds in the aromatic rings and substituent positions that facilitates their identifica‐ tion and the development of spectrometric analytical techniques is easily accomplished. In this sense, spectrophotometric methods are more practical, reproducible and inexpensive than other techniques and are therefore favoured for the development of analytical method‐

In addition to producing compounds such as carbohydrates, lipids, proteins and nucleic acids directly involved in their essential growth functions, plants have an arsenal of enzymes capable

distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Cavalcanti de Amorim et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Cavalcanti de Amorim et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

## **Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained in Plant Samples**

Elba Lúcia Cavalcanti de Amorim, Valérium Thijan Nobre de Almeida de Castro, Joabe Gomes de Melo, Allan Jonathan Chernichiarro Corrêa and Tadeu José da Silva Peixoto Sobrinho

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51686

**1. Introduction**

The quality control of raw materials and products from plants is one of the topics most discussed by universities and health surveillance agencies. One of the primary tools used to ensure the reliability of production processes is the use of Standard Operating Procedures (SOPs). SOPs sequentially describe the steps of a particular methodology so that it can be reproduced by different analysts, which minimises variations in their implementation and improves the standardisation of the final product.

Several techniques, such as high performance liquid chromatography, gas chromatography and mass spectrometry, can be used in SOPs to control the quality of plant phenolic com‐ pounds [1,2]. However, these compounds have a characteristic spectrum produced by the double bonds in the aromatic rings and substituent positions that facilitates their identifica‐ tion and the development of spectrometric analytical techniques is easily accomplished. In this sense, spectrophotometric methods are more practical, reproducible and inexpensive than other techniques and are therefore favoured for the development of analytical method‐ ologies for such determinations.

In addition to producing compounds such as carbohydrates, lipids, proteins and nucleic acids directly involved in their essential growth functions, plants have an arsenal of enzymes capable

© 2012 Cavalcanti de Amorim et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Cavalcanti de Amorim et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of producing, processing and accumulating several other substances not necessarily related to the maintenance of their life [3]. All of these reactions can be defined as secondary metab‐ olism, the products of which provide advantages for both survival and species perpetuation in the plant's ecosystem [4]. However, this protection has a cost for the plant because meta‐ bolic resources that could increase its biomass are used to produce these compounds. In addition to protection, secondary metabolites perform important ecological functions such as inhibiting the germination and growth of other plants, attracting both pollinators and seeddispersing animals and providing chemical defences against microorganisms [5].

Condensed tannins, also known as proanthocyanidins (Figure 2), can contain dozens of units of flavan-3-ols (catechin) or flavan-3,4-diols (leucoanthocyanidins). These units have a complex structure and are resistant to hydrolysis; however, they can be soluble in aqueous

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

in Plant Samples

49

http://dx.doi.org/10.5772/51686

Hydrolysable and condensed tannins may occur in the same plant simultaneously. Howev‐ er, the hydrolysable tannins are characteristic of Magnoliopsida herbaceous and woody plants and are restricted to certain taxonomic families. Ellagitannins have been used as taxonomic markers, particularly for Hemamelidaceae, Dilenidaceaa and Rosaceae. Condensed tannins have been identified in all plant groups, including Gymnosperms and Pteridophytes [10]. These secondary metabolites were initially identified by their astringent taste and capacity to bind proteins, which allows for the precipitation and formation of complexes with colla‐ gen skin fibres to increase their resistance to water and heat. Chemically speaking, hydro‐ phobic interactions and hydrogen bonds between the phenolic groups in tannins and some macromolecules explain these features. However, the stability of the formed complexes only results after the formation of covalent bonds via the oxidation of tannins by quinones [11].

Since antiquity, plants containing tannins have been used medicinally as anti-inflamma‐ tory, antimicrobial, antitumor and antiviral agents and to treat both wound sand burns [7]. Tannins are also used to manufacture beverages and process animal skin into leath‐ er. Some researchers have shown that tannins protect plants against attack by herbi‐

Although the use of tannin in the tanning industry has become restricted, interest in study‐ ing the ingestion of foods containing tannins to prevent diseases such as atherosclerosis or certain types of cancer has increased because of various epidemiological studies. Some stud‐ ies report that the complexation of tannins with proteins gives them an important role in controlling bacteria, fungi and insects [13-15]. Other studies examined the inhibitory action of the enzyme reverse transcriptase [16] and the anticarcinogenic activity associated with

organic solvents because of their structure [7].

**Figure 2.** Basic structures of catechin and proanthocyanidins (condensed tannin).

vores and pathogens [12].

Phenolic compounds, which have one or more hydroxyl groups linked to an aromatic ring, stand out from other classes of plant secondary metabolites because they are widely distrib‐ uted and have various ecological functions that are scientifically proven to have numerous pharmacological activities and are well represented by tannins, flavonoids and coumarins.

#### **1.1. Tannins**

Tannins are water soluble phenolic compounds with a molecular weight between 500 and 3000 Daltons and may be chemically classified into two groups: hydrolysable tannins and condensed tannins [6,7]. Hydrolysable tannins are connected by ester-carboxyl linkages, which undergo hydrolysis under acidic and basic conditions [8]. Figure 1 presents an exam‐ ple of hydrolysable tannin (gallotannin), connected through a polyol (usually β-D-glucose) with the hydroxyl group esterified by gallic acid. Polyphenols connected with ellagic acid are called ellagitannins [9].

**Figure 1.** Structure of gallic acid, ellagic acid and 1,3,6-tri-O-galloyl-β-D-glucose, a hydrolysable tannin.

Condensed tannins, also known as proanthocyanidins (Figure 2), can contain dozens of units of flavan-3-ols (catechin) or flavan-3,4-diols (leucoanthocyanidins). These units have a complex structure and are resistant to hydrolysis; however, they can be soluble in aqueous organic solvents because of their structure [7].

**Figure 2.** Basic structures of catechin and proanthocyanidins (condensed tannin).

of producing, processing and accumulating several other substances not necessarily related to the maintenance of their life [3]. All of these reactions can be defined as secondary metab‐ olism, the products of which provide advantages for both survival and species perpetuation in the plant's ecosystem [4]. However, this protection has a cost for the plant because meta‐ bolic resources that could increase its biomass are used to produce these compounds. In addition to protection, secondary metabolites perform important ecological functions such as inhibiting the germination and growth of other plants, attracting both pollinators and seed-

Phenolic compounds, which have one or more hydroxyl groups linked to an aromatic ring, stand out from other classes of plant secondary metabolites because they are widely distrib‐ uted and have various ecological functions that are scientifically proven to have numerous pharmacological activities and are well represented by tannins, flavonoids and coumarins.

Tannins are water soluble phenolic compounds with a molecular weight between 500 and 3000 Daltons and may be chemically classified into two groups: hydrolysable tannins and condensed tannins [6,7]. Hydrolysable tannins are connected by ester-carboxyl linkages, which undergo hydrolysis under acidic and basic conditions [8]. Figure 1 presents an exam‐ ple of hydrolysable tannin (gallotannin), connected through a polyol (usually β-D-glucose) with the hydroxyl group esterified by gallic acid. Polyphenols connected with ellagic acid

dispersing animals and providing chemical defences against microorganisms [5].

**Figure 1.** Structure of gallic acid, ellagic acid and 1,3,6-tri-O-galloyl-β-D-glucose, a hydrolysable tannin.

**1.1. Tannins**

48 Latest Research into Quality Control

are called ellagitannins [9].

Hydrolysable and condensed tannins may occur in the same plant simultaneously. Howev‐ er, the hydrolysable tannins are characteristic of Magnoliopsida herbaceous and woody plants and are restricted to certain taxonomic families. Ellagitannins have been used as taxonomic markers, particularly for Hemamelidaceae, Dilenidaceaa and Rosaceae. Condensed tannins have been identified in all plant groups, including Gymnosperms and Pteridophytes [10].

These secondary metabolites were initially identified by their astringent taste and capacity to bind proteins, which allows for the precipitation and formation of complexes with colla‐ gen skin fibres to increase their resistance to water and heat. Chemically speaking, hydro‐ phobic interactions and hydrogen bonds between the phenolic groups in tannins and some macromolecules explain these features. However, the stability of the formed complexes only results after the formation of covalent bonds via the oxidation of tannins by quinones [11].

Since antiquity, plants containing tannins have been used medicinally as anti-inflamma‐ tory, antimicrobial, antitumor and antiviral agents and to treat both wound sand burns [7]. Tannins are also used to manufacture beverages and process animal skin into leath‐ er. Some researchers have shown that tannins protect plants against attack by herbi‐ vores and pathogens [12].

Although the use of tannin in the tanning industry has become restricted, interest in study‐ ing the ingestion of foods containing tannins to prevent diseases such as atherosclerosis or certain types of cancer has increased because of various epidemiological studies. Some stud‐ ies report that the complexation of tannins with proteins gives them an important role in controlling bacteria, fungi and insects [13-15]. Other studies examined the inhibitory action of the enzyme reverse transcriptase [16] and the anticarcinogenic activity associated with green tea and diets rich in fruits containing tannins [17]. It is generally believed that the pharmacological activity of tannins occurs via their complexation with metal ions, antioxi‐ dant activity or the ability to complex with macromolecules.

medicine as a hypoglycemic, diuretic and antihypertensive agent [31,32]. Several plantbased products are sold in open markets, pharmacies and natural product stores [33]. Though not officially recommended, herbal products from the leaves of pata-de-vaca (*Bauhi‐ nia* L.) are popularly used for therapeutic purposes. This plant was used in our study as a

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

in Plant Samples

51

http://dx.doi.org/10.5772/51686

Coumarins are lactones of *O*-hydroxy-cinnamic acid derived from trans-cinnamic acid via oxidation-reduction and isomerisation to produce 1,2-benzopyrone. Coumarins are divided into simple coumarins, furanocoumarins, pyranocoumarins, dimeric coumarins and chro‐ mones (Figure 4) [34]. The difference between these classes is the position of the radical in the coumarin lactone ring, which varies between C-6, C-7 and C-8. Chromones represent a group of natural isomers to coumarin that may be linearly and angularly prenylated at C-6

Coumarins are used as antioxidants, anti-HIV drugs, antispasmodics, spasmolytics, hypoli‐ pidemics, hypotensives and vasodilating agents [34]; however, they are also used in food flavouring, perfumes, tobacco and cosmetic products [35]. It is estimated that the daily hu‐

Coumarins are a class of secondary metabolites widely distributed throughout the Plantae kingdom and found in Fungi and Bacteria as well [37]. In plants, coumarins are found fre‐ quently in the families of Apiaceae, Rutaceae and Asteraceae, and less frequently in the fam‐

man exposure to coumarins from cosmetics and perfumes is 0.04 mg/kg/day [36].

ilies of Fabaceae, Oleaceae, Moraceae and Thymeleaceae [38].

and C-8, respectively, in furanochromones and pyranochromones, respectively [34].

reference species for flavonoids.

**Figure 4.** Basic structure of coumarins and chromones.

**1.3. Coumarins**

*Maytenus ilicifolia* (Schrad.) Planch. (Celastraceae), popularly known as "espinheira-santa", is a species native to Brazil traditionally used to treat digestive disorders and the literature cites tannins as compounds that act to protect the stomach by helping to treat ulcers and gastritis [18-20]. In addition to these activities, *M. ilicifolia* has other pharmacological appli‐ cations, such as anticancer, antimicrobial and antioxidant activity and treatment of the cen‐ tral nervous system [21]. For these reasons, *M. ilicifolia* was chosen as a reference species in this study.

#### **1.2. Flavonoids**

Flavonoids comprise a group of natural substances with great structural diversity and there are currently more than nine thousand known flavonoids that do not occur in humans but can be found in various plant parts such as the leaves, fruits, bark, roots, stems and flowers [22,23]. Flavonoids (Figure 3) are composed of a simple skeleton containing two phenol rings connected by a propionic chain; where ring A is the acetate derivative and both ring B and the three-carbon bridge are derived via a shikimate pathway, which may be associated with carbohydrates (heterosides), un associated (aglycones) or polymerised further (antho‐ cyanins) [24].

**Figure 3.** Basic structure of flavonoids.

This class of metabolites has several biological functions, such as defence against both herbi‐ vores and pathogens, the perpetuation of the species by attracting seed dispersing animals, protection from ultraviolet rays and allelopathy [24,25]. Flavonoids also possess important pharmacological properties, such as antioxidant, antiinflammatory, anti-thrombogenic, anti‐ microbial, anticancer, antidiabetic and hypocholesterolemic activities [23,26].

Studies show that flavonoids are chemical markers responsible for various pharmacological activities performed by the genus *Bauhinia* [27-29]. *Bauhinia forficata* Link (Fabaceae), popu‐ larly known as "pata-de-vaca", is a tree native to Brazil that prevails in the phytogeographi‐ cal area of Mata Atlântica [30] and can also be found in Cerrado. Pata-de-vaca is used in folk medicine as a hypoglycemic, diuretic and antihypertensive agent [31,32]. Several plantbased products are sold in open markets, pharmacies and natural product stores [33]. Though not officially recommended, herbal products from the leaves of pata-de-vaca (*Bauhi‐ nia* L.) are popularly used for therapeutic purposes. This plant was used in our study as a reference species for flavonoids.

#### **1.3. Coumarins**

green tea and diets rich in fruits containing tannins [17]. It is generally believed that the pharmacological activity of tannins occurs via their complexation with metal ions, antioxi‐

*Maytenus ilicifolia* (Schrad.) Planch. (Celastraceae), popularly known as "espinheira-santa", is a species native to Brazil traditionally used to treat digestive disorders and the literature cites tannins as compounds that act to protect the stomach by helping to treat ulcers and gastritis [18-20]. In addition to these activities, *M. ilicifolia* has other pharmacological appli‐ cations, such as anticancer, antimicrobial and antioxidant activity and treatment of the cen‐ tral nervous system [21]. For these reasons, *M. ilicifolia* was chosen as a reference species in

Flavonoids comprise a group of natural substances with great structural diversity and there are currently more than nine thousand known flavonoids that do not occur in humans but can be found in various plant parts such as the leaves, fruits, bark, roots, stems and flowers [22,23]. Flavonoids (Figure 3) are composed of a simple skeleton containing two phenol rings connected by a propionic chain; where ring A is the acetate derivative and both ring B and the three-carbon bridge are derived via a shikimate pathway, which may be associated with carbohydrates (heterosides), un associated (aglycones) or polymerised further (antho‐

This class of metabolites has several biological functions, such as defence against both herbi‐ vores and pathogens, the perpetuation of the species by attracting seed dispersing animals, protection from ultraviolet rays and allelopathy [24,25]. Flavonoids also possess important pharmacological properties, such as antioxidant, antiinflammatory, anti-thrombogenic, anti‐

Studies show that flavonoids are chemical markers responsible for various pharmacological activities performed by the genus *Bauhinia* [27-29]. *Bauhinia forficata* Link (Fabaceae), popu‐ larly known as "pata-de-vaca", is a tree native to Brazil that prevails in the phytogeographi‐ cal area of Mata Atlântica [30] and can also be found in Cerrado. Pata-de-vaca is used in folk

microbial, anticancer, antidiabetic and hypocholesterolemic activities [23,26].

dant activity or the ability to complex with macromolecules.

this study.

**1.2. Flavonoids**

50 Latest Research into Quality Control

cyanins) [24].

**Figure 3.** Basic structure of flavonoids.

Coumarins are lactones of *O*-hydroxy-cinnamic acid derived from trans-cinnamic acid via oxidation-reduction and isomerisation to produce 1,2-benzopyrone. Coumarins are divided into simple coumarins, furanocoumarins, pyranocoumarins, dimeric coumarins and chro‐ mones (Figure 4) [34]. The difference between these classes is the position of the radical in the coumarin lactone ring, which varies between C-6, C-7 and C-8. Chromones represent a group of natural isomers to coumarin that may be linearly and angularly prenylated at C-6 and C-8, respectively, in furanochromones and pyranochromones, respectively [34].

**Figure 4.** Basic structure of coumarins and chromones.

Coumarins are used as antioxidants, anti-HIV drugs, antispasmodics, spasmolytics, hypoli‐ pidemics, hypotensives and vasodilating agents [34]; however, they are also used in food flavouring, perfumes, tobacco and cosmetic products [35]. It is estimated that the daily hu‐ man exposure to coumarins from cosmetics and perfumes is 0.04 mg/kg/day [36].

Coumarins are a class of secondary metabolites widely distributed throughout the Plantae kingdom and found in Fungi and Bacteria as well [37]. In plants, coumarins are found fre‐ quently in the families of Apiaceae, Rutaceae and Asteraceae, and less frequently in the fam‐ ilies of Fabaceae, Oleaceae, Moraceae and Thymeleaceae [38].

*Mikania glomerata* Spreng. (Asteraceae), popularly known in Brazil as "guaco", has existed in the Brazilian Pharmacopoeia since 1929 and is used as an expectorant to treat respiratory problems. Most herbal products are marketed as some form of syrup; however, they are produced from fluid extracts and tinctures [39,40]. Chemical studies of this species show coumarins to be a major component that can be used as a chemical marker for the quality control of products based on guaco [41-43].

**3.1. Standard operating procedure for the quantification of tannins**

calculated as the difference between the total and waste phenol content.

or more molecules of flavan-3-ol or flavan-3,4-diol [3] [7].

**Reagents:**

distilled water.

tion of 1 mg/mL.

tannic acid concentration will be 1-5 μg/mL.

**Description:** Tannins are phenolic compounds with the ability to bind macromolecules, es‐ pecially proteins. This class of compounds has traditionally been divided into two groups: hydrolysable and condensed tannins. Hydrolysable tannins are characterised by a central polyol, usually β-D-glucose, containing hydroxy groups esterified with gallic acid and ella‐ gic acid. Condensed tannins are oligomers or polymers formed by the condensation of two

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

in Plant Samples

53

http://dx.doi.org/10.5772/51686

**Principle of the method:** The phenolic compounds in the sample are oxidised using the Folin-Ciocalteu reagent. This reagent is a mixture of phosphotungstic and phosphomolybdic acids that are reduced by the oxidation of phenolic compounds in a mixture of tungsten and molybdenum oxides. The blue colour produced by the oxides has a maximum absorption at 760 nm and is proportional to the total phenolic concentration [47]. The tannin content is

(1) Tannic acid (0.1 mg/mL, w/v): Dissolve 10 mg of tannic acid in 100 ml of distilled water.

(2) Folin-Ciocalteu reagent (10%, v/v): Dilute 5 ml of Folin-Ciocalteu reagent with 45 mL of

(3) Sodium carbonate Na2CO3 (7.5%, w/v): Dissolve 7.5 g of Na2CO3 in 100 ml of distilled

**Preparation of samples:** The powdered sample (500 mg) should be extracted with 50 mL of 80% methanol for 30 minutes on a hot plate. The extract should be filtered through filter pa‐ per into a 50 mL volumetric flask and the volume should be completed using the same sol‐ vent. The final extract concentration will be 10 mg/mL. If the products are liquid, they must be evaporated. The dried extract should be dissolved in 80% methanol for a final concentra‐

**Calibration curve:** The calibration curve must be prepared using 100-500 μL aliquots of the tannic acid solution, 500 μL of the Folin-Ciocalteu solution and 1 mL of the sodium carbo‐ nate solution. The final volume should be adjusted to 10 mL with distilled water. The final

**Measurement procedure:** To quantify the total phenol concentration (that is, all of the phe‐ nols present in the sample), 500 μL of the extract must be transferred to a test tube. Next, 500 μL of the Folin-Ciocalteu solution, 1 mL of the sodium carbonate solution and 8 ml of distil‐ led water are added. The samples remain at room temperature for 30 minutes. The spectro‐ photometer should be adjusted to a wavelength of 760 nm and the equipment must be rinsed with distilled water. To quantify the phenol waste, (i.e., the phenols present in the sample except for the proteins precipitated with the tannin) 500 mg of casein is weighed and

water. If necessary, solubilising the solution on a heating plate and magnetic stirrer.

(4) Methanol (80%, v/v): Dilute 800 ml of methanol with 200 ml of distilled water.

The reagent volume is sufficient to examine a maximum of 100 analyses.

Coumarins have a characteristic UV spectrum due to the nature and position of their sub‐ stituents, which facilitates both their identification and the development of analytical spec‐ trophotometric techniques [11]. For these reasons, *M. glomerata* was chosen as the reference species to quantify the coumarin level.

#### **2. Problem statement**

Despite extensive literature presenting various analytical methods, the development of an SOP is often difficult for three reasons: 1) the work does not detail the difficulties and adjust‐ ments required to implement the methodology, 2) the steps are not clearly presented for re‐ production and 3) the limits of interpretation are not discussed. One criterion recommended by the National Sanitary Surveillance Agency (Agência Nacional de Vigilância Sanitária - ANVISA) in Brazil for the standardisation of herbal drugs is the active compound content or chemical class, which is the total concentration of tannins for products based on *M. ilicifolia* (espinheira-santa) [44], flavonoids for *B. forficata* (pata-de-vaca) [45,46] and coumarins for *M. glomerata* (guaco) [44].

Thus, this paper presents research protocols adopted by our research group to study the lev‐ els of tannins, flavonoids and coumarins from plant extracts and the experimental applica‐ tion of these SOPs to analyse products sold in markets (pharmacies and natural product stores) as phytomedicines1 or plant drugs2 with high commercial value.

#### **3. Standard operating procedures (POP)**

The following SOPs describe the chemical classes to be analysed and the chemical basis of the methods. They provide a detailed list of all the reagents required for the preparation and describe the experimental procedure to be followed. Finally, there is a list of references used in the development of the SOP.

1 "All medicine is obtained using solely active raw vegetables. It is characterised by knowledge of the effectiveness and risks of their use, as well as the reproducibility and consistency of its quality. Its efficacy and safety are validated through ethnopharmacological surveys of use, documentation, technical and scientific publications or clinical trial phase 3"[44].

2 "Medicinal plant or their parts, after collection processes, stabilisation and drying and can be full, erasures, crushed or powdered" [44].

#### **3.1. Standard operating procedure for the quantification of tannins**

**Description:** Tannins are phenolic compounds with the ability to bind macromolecules, es‐ pecially proteins. This class of compounds has traditionally been divided into two groups: hydrolysable and condensed tannins. Hydrolysable tannins are characterised by a central polyol, usually β-D-glucose, containing hydroxy groups esterified with gallic acid and ella‐ gic acid. Condensed tannins are oligomers or polymers formed by the condensation of two or more molecules of flavan-3-ol or flavan-3,4-diol [3] [7].

**Principle of the method:** The phenolic compounds in the sample are oxidised using the Folin-Ciocalteu reagent. This reagent is a mixture of phosphotungstic and phosphomolybdic acids that are reduced by the oxidation of phenolic compounds in a mixture of tungsten and molybdenum oxides. The blue colour produced by the oxides has a maximum absorption at 760 nm and is proportional to the total phenolic concentration [47]. The tannin content is calculated as the difference between the total and waste phenol content.

#### **Reagents:**

*Mikania glomerata* Spreng. (Asteraceae), popularly known in Brazil as "guaco", has existed in the Brazilian Pharmacopoeia since 1929 and is used as an expectorant to treat respiratory problems. Most herbal products are marketed as some form of syrup; however, they are produced from fluid extracts and tinctures [39,40]. Chemical studies of this species show coumarins to be a major component that can be used as a chemical marker for the quality

Coumarins have a characteristic UV spectrum due to the nature and position of their sub‐ stituents, which facilitates both their identification and the development of analytical spec‐ trophotometric techniques [11]. For these reasons, *M. glomerata* was chosen as the reference

Despite extensive literature presenting various analytical methods, the development of an SOP is often difficult for three reasons: 1) the work does not detail the difficulties and adjust‐ ments required to implement the methodology, 2) the steps are not clearly presented for re‐ production and 3) the limits of interpretation are not discussed. One criterion recommended by the National Sanitary Surveillance Agency (Agência Nacional de Vigilância Sanitária - ANVISA) in Brazil for the standardisation of herbal drugs is the active compound content or chemical class, which is the total concentration of tannins for products based on *M. ilicifolia* (espinheira-santa) [44], flavonoids for *B. forficata* (pata-de-vaca) [45,46] and coumarins for *M.*

Thus, this paper presents research protocols adopted by our research group to study the lev‐ els of tannins, flavonoids and coumarins from plant extracts and the experimental applica‐ tion of these SOPs to analyse products sold in markets (pharmacies and natural product

The following SOPs describe the chemical classes to be analysed and the chemical basis of the methods. They provide a detailed list of all the reagents required for the preparation and describe the experimental procedure to be followed. Finally, there is a list of references used

1 "All medicine is obtained using solely active raw vegetables. It is characterised by knowledge of the effectiveness and risks of their use, as well as the reproducibility and consistency of its quality. Its efficacy and safety are validated through ethnopharmacological surveys of use, documentation, technical and scientific publications or clinical trial

2 "Medicinal plant or their parts, after collection processes, stabilisation and drying and can be full, erasures, crushed

with high commercial value.

or plant drugs2

**3. Standard operating procedures (POP)**

control of products based on guaco [41-43].

species to quantify the coumarin level.

**2. Problem statement**

52 Latest Research into Quality Control

*glomerata* (guaco) [44].

stores) as phytomedicines1

in the development of the SOP.

phase 3"[44].

or powdered" [44].

(1) Tannic acid (0.1 mg/mL, w/v): Dissolve 10 mg of tannic acid in 100 ml of distilled water.

(2) Folin-Ciocalteu reagent (10%, v/v): Dilute 5 ml of Folin-Ciocalteu reagent with 45 mL of distilled water.

(3) Sodium carbonate Na2CO3 (7.5%, w/v): Dissolve 7.5 g of Na2CO3 in 100 ml of distilled water. If necessary, solubilising the solution on a heating plate and magnetic stirrer.

(4) Methanol (80%, v/v): Dilute 800 ml of methanol with 200 ml of distilled water.

The reagent volume is sufficient to examine a maximum of 100 analyses.

**Preparation of samples:** The powdered sample (500 mg) should be extracted with 50 mL of 80% methanol for 30 minutes on a hot plate. The extract should be filtered through filter pa‐ per into a 50 mL volumetric flask and the volume should be completed using the same sol‐ vent. The final extract concentration will be 10 mg/mL. If the products are liquid, they must be evaporated. The dried extract should be dissolved in 80% methanol for a final concentra‐ tion of 1 mg/mL.

**Calibration curve:** The calibration curve must be prepared using 100-500 μL aliquots of the tannic acid solution, 500 μL of the Folin-Ciocalteu solution and 1 mL of the sodium carbo‐ nate solution. The final volume should be adjusted to 10 mL with distilled water. The final tannic acid concentration will be 1-5 μg/mL.

**Measurement procedure:** To quantify the total phenol concentration (that is, all of the phe‐ nols present in the sample), 500 μL of the extract must be transferred to a test tube. Next, 500 μL of the Folin-Ciocalteu solution, 1 mL of the sodium carbonate solution and 8 ml of distil‐ led water are added. The samples remain at room temperature for 30 minutes. The spectro‐ photometer should be adjusted to a wavelength of 760 nm and the equipment must be rinsed with distilled water. To quantify the phenol waste, (i.e., the phenols present in the sample except for the proteins precipitated with the tannin) 500 mg of casein is weighed and transferred into a 25 ml Erlenmeyer flask before adding 5 ml of the extract and 5 ml of water distilled. After two hours (time required for the complexing of the tannins to the total pro‐ tein), the extracts are filtered into a 10 mL volumetric flasks and its volume is adjusted with distilled water. The phenols are considered to be equal to the residue from the total phenol. These assays are performed at least in triplicate and the total phenolic content is expressed as milligrams of tannic acid equivalents per gram of sample or extract (mg TAE/g).

**Calibration curve:** The calibration curve must be prepared using 100-1000 μL aliquots of the rutin solution, 500 μL of the acetic acid solution, 2 mL of the pyridine solution and 1 ml of the reagent aluminium chloride solution. The final volume should be adjusted to 10 mL with

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

in Plant Samples

55

http://dx.doi.org/10.5772/51686

**Measurement procedure:** To quantify the flavonoids, 500 μL of the extract should be trans‐ ferred to a test tube. Next, 500 μL of the acetic acid solution, 2 mL of the pyridine solution, 1 ml of the reagent aluminium chloride solution and 6 ml of 80% methanol will be added. The samples remain at room temperature for 30 minutes. The spectrophotometer should be ad‐ justed to a wavelength of 420 nm and the equipment must be rinsed with distilled water. The test shall be performed at least in triplicate and the flavonoid content is expressed as

Peixoto Sobrinho T. J. S, Silva C. H. T. P., Nascimento J. E., Monteiro J. M., Albuquerque U. P., Amorim E. L. C. Validação de metodologia espectrofotométrica para quantificação dos flavonóides de *Bauhinia cheilantha* (Bongard) Steudel. Brazilian Journal of Pharmaceutical

Zuanazzi, J. A. S.; Montanha, J. A. Flavonóides. In: Simões C. M. O., Schenkel E. P., Gos‐ manm, G, Mello J. C. P., Mentz L. A., Petrovick P. R. (ed.) Farmacognosia: da planta ao med‐ icamento. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2004, p.577-614 [24].

**Description:** Coumarins are lactones of *O*-hydroxy-cinnamic acid and are metabolites of phenylalanine. 1,2-benzopyrone is the simplest coumarin representative, others being furo‐

**Principle of the method:** The Borntrager reaction is based on the solubility of free coumarin derivatives in polar organic solvents and the solubility of their soluble alkali phenolates. Coumarin absorbs at 280 nm; however, ionisation of phenolic hydroxyls in the molecule by alkaline hydroxide causes a bathochromic deviation to 320 nm, which is proportional to the

(1) 1,2-benzopyrone (1 mg/mL, w/v): Dissolve 10 mg of coumarin in 10 ml of distilled water.

(3) Hydrochloric acid solution, HCl (0.1 M, v/v): Dilute 10 ml of concentrated hydrochloric

**Preparation of samples:** The powdered sample (500 mg) should be extracted with 50 mL of 80% methanol for 30 minutes on a hot plate. The extract should be filtered through filter paper

(2) Lead acetate (5%, w/v): Dissolve 2,5 g of lead acetate in 50 ml of distilled water.

(4) Methanol (80%, v/v): Dilute 80 ml of methanol with 20 ml of distilled water.

The reagent volume is sufficient to examine a maximum of 100 analyses.

80% methanol. The final rutin concentration will be 1-10 μg/mL.

milligrams of rutin equivalents per gram of sample or extract (mg RE/g).

**3.3. Standard operating procedure for the quantification of coumarins**

coumarins, pyranocoumarins, dimeric coumarins and chromones [34].

**References:**

Sciences 2008; 44 (4) 683-689 [48].

coumarin concentration [40].

acid with 1000 ml of distilled water.

**Reagents:**

#### **References:**

Amorim E. L. C, Nascimento J. E., Monteiro J. M., Peixoto Sobrinho T. J. S, Araújo T. A. S., Albuquerque U. P. A simple and accurate procedure for the determination of tannin and fla‐ vonoid levels and some applications in ethnobotany and ethnopharmacology. Functional Ecosystems and Communities 2008; 2(1) 88-94 [47].

Santos S. C., Mello J. C. P. Taninos. In: Simões C. M. O., Schenkel E. P., Gosmanm, G., Mello J. C. P., Mentz L. A., Petrovick P. R. (ed.) Farmacognosia: da planta ao medicamento. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2004. p.615-656 [3].

#### **3.2. Standard operating procedure for the quantification of flavonoids**

**Description:** Flavonoids are the most important and diverse phenolic compounds. Most fla‐ vonoids have 15 carbon atoms and consist of two phenol rings connected by a chain of three carbons to form a tricyclic compound [24].

**Principle of the method:** The aluminium ion (Al3+) is reacted with the flavonoids in the sam‐ ple to form the stable flavonoid-Al3+complex, which has a yellow colour and intensity pro‐ portional to the flavonoid concentration. This reaction causes a bathochromic shift and intensification in the absorption, which can be measured without influence from other phe‐ nolic compounds present in the sample [48].

#### **Reagents:**

(1) Rutin (0.1 mg/mL, w/v): Dissolve 10 mg of rutin in 100 ml of methanol.

(2) Acetic acid solution (60%, v/v): Dilute 30 ml of acetic acid with 20 ml of methanol.

(3) Pyridine Solution (20%, v/v): Dilute 40 ml of pyridine with 160 ml of methanol.

(4) Aluminium chloride solution AlCl3 (5%, w/v): Dissolve 5 g AlCl3 in 100 mL of methanol. If necessary, complete dissolution via magnetic stirring.

(5) Methanol (80%, v/v). Dilute 80 ml of methanol with 20 ml of distilled water.

The reagent volume is sufficient to examine a maximum of 100 analyses.

**Preparation of samples:** The powdered sample (500 mg) should be extracted with 50 mL of 80% methanol on a hot plate for 30 minutes. The extract should be filtered through filter paper into a 50 mL volumetric flask and the volume should be adjusted with the same solvent. The final extract concentration will be 10 mg/mL. Liquid products must be evaporated. The dried extract should be dissolved in 80% methanol to obtain a final concentration of 1 mg/mL.

**Calibration curve:** The calibration curve must be prepared using 100-1000 μL aliquots of the rutin solution, 500 μL of the acetic acid solution, 2 mL of the pyridine solution and 1 ml of the reagent aluminium chloride solution. The final volume should be adjusted to 10 mL with 80% methanol. The final rutin concentration will be 1-10 μg/mL.

**Measurement procedure:** To quantify the flavonoids, 500 μL of the extract should be trans‐ ferred to a test tube. Next, 500 μL of the acetic acid solution, 2 mL of the pyridine solution, 1 ml of the reagent aluminium chloride solution and 6 ml of 80% methanol will be added. The samples remain at room temperature for 30 minutes. The spectrophotometer should be ad‐ justed to a wavelength of 420 nm and the equipment must be rinsed with distilled water. The test shall be performed at least in triplicate and the flavonoid content is expressed as milligrams of rutin equivalents per gram of sample or extract (mg RE/g).

#### **References:**

transferred into a 25 ml Erlenmeyer flask before adding 5 ml of the extract and 5 ml of water distilled. After two hours (time required for the complexing of the tannins to the total pro‐ tein), the extracts are filtered into a 10 mL volumetric flasks and its volume is adjusted with distilled water. The phenols are considered to be equal to the residue from the total phenol. These assays are performed at least in triplicate and the total phenolic content is expressed

Amorim E. L. C, Nascimento J. E., Monteiro J. M., Peixoto Sobrinho T. J. S, Araújo T. A. S., Albuquerque U. P. A simple and accurate procedure for the determination of tannin and fla‐ vonoid levels and some applications in ethnobotany and ethnopharmacology. Functional

Santos S. C., Mello J. C. P. Taninos. In: Simões C. M. O., Schenkel E. P., Gosmanm, G., Mello J. C. P., Mentz L. A., Petrovick P. R. (ed.) Farmacognosia: da planta ao medicamento. Porto

**Description:** Flavonoids are the most important and diverse phenolic compounds. Most fla‐ vonoids have 15 carbon atoms and consist of two phenol rings connected by a chain of three

**Principle of the method:** The aluminium ion (Al3+) is reacted with the flavonoids in the sam‐ ple to form the stable flavonoid-Al3+complex, which has a yellow colour and intensity pro‐ portional to the flavonoid concentration. This reaction causes a bathochromic shift and intensification in the absorption, which can be measured without influence from other phe‐

as milligrams of tannic acid equivalents per gram of sample or extract (mg TAE/g).

Alegre: Universidade Federal do Rio Grande do Sul; 2004. p.615-656 [3].

**3.2. Standard operating procedure for the quantification of flavonoids**

(1) Rutin (0.1 mg/mL, w/v): Dissolve 10 mg of rutin in 100 ml of methanol.

(2) Acetic acid solution (60%, v/v): Dilute 30 ml of acetic acid with 20 ml of methanol.

(4) Aluminium chloride solution AlCl3 (5%, w/v): Dissolve 5 g AlCl3 in 100 mL of methanol.

**Preparation of samples:** The powdered sample (500 mg) should be extracted with 50 mL of 80% methanol on a hot plate for 30 minutes. The extract should be filtered through filter paper into a 50 mL volumetric flask and the volume should be adjusted with the same solvent. The final extract concentration will be 10 mg/mL. Liquid products must be evaporated. The dried extract should be dissolved in 80% methanol to obtain a final concentration of 1 mg/mL.

(3) Pyridine Solution (20%, v/v): Dilute 40 ml of pyridine with 160 ml of methanol.

(5) Methanol (80%, v/v). Dilute 80 ml of methanol with 20 ml of distilled water.

The reagent volume is sufficient to examine a maximum of 100 analyses.

Ecosystems and Communities 2008; 2(1) 88-94 [47].

carbons to form a tricyclic compound [24].

nolic compounds present in the sample [48].

If necessary, complete dissolution via magnetic stirring.

**References:**

54 Latest Research into Quality Control

**Reagents:**

Peixoto Sobrinho T. J. S, Silva C. H. T. P., Nascimento J. E., Monteiro J. M., Albuquerque U. P., Amorim E. L. C. Validação de metodologia espectrofotométrica para quantificação dos flavonóides de *Bauhinia cheilantha* (Bongard) Steudel. Brazilian Journal of Pharmaceutical Sciences 2008; 44 (4) 683-689 [48].

Zuanazzi, J. A. S.; Montanha, J. A. Flavonóides. In: Simões C. M. O., Schenkel E. P., Gos‐ manm, G, Mello J. C. P., Mentz L. A., Petrovick P. R. (ed.) Farmacognosia: da planta ao med‐ icamento. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2004, p.577-614 [24].

#### **3.3. Standard operating procedure for the quantification of coumarins**

**Description:** Coumarins are lactones of *O*-hydroxy-cinnamic acid and are metabolites of phenylalanine. 1,2-benzopyrone is the simplest coumarin representative, others being furo‐ coumarins, pyranocoumarins, dimeric coumarins and chromones [34].

**Principle of the method:** The Borntrager reaction is based on the solubility of free coumarin derivatives in polar organic solvents and the solubility of their soluble alkali phenolates. Coumarin absorbs at 280 nm; however, ionisation of phenolic hydroxyls in the molecule by alkaline hydroxide causes a bathochromic deviation to 320 nm, which is proportional to the coumarin concentration [40].

#### **Reagents:**

(1) 1,2-benzopyrone (1 mg/mL, w/v): Dissolve 10 mg of coumarin in 10 ml of distilled water.

(2) Lead acetate (5%, w/v): Dissolve 2,5 g of lead acetate in 50 ml of distilled water.

(3) Hydrochloric acid solution, HCl (0.1 M, v/v): Dilute 10 ml of concentrated hydrochloric acid with 1000 ml of distilled water.

(4) Methanol (80%, v/v): Dilute 80 ml of methanol with 20 ml of distilled water.

The reagent volume is sufficient to examine a maximum of 100 analyses.

**Preparation of samples:** The powdered sample (500 mg) should be extracted with 50 mL of 80% methanol for 30 minutes on a hot plate. The extract should be filtered through filter paper into a 50 mL volumetric flask and the final volume should be adjusted with the same sol‐ vent. The final extract concentration will be 10 mg/mL. Liquid products must be evaporated. The dried extract should be dissolved in 80% methanol for a final concentration of 1 mg/mL.

Analysis of the active component concentrations were conducted as listed in the described

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

To quantify the active components, calibration curves with increasing concentrations pro‐ portional to their absorbance were constructed. A correlation equation was obtained from these curves (generally linear) of the type *y = ax + b*, where *y* corresponds to the absorbance of the sample and *x* the concentration. To convert the absorbance values (nm) to sample concentration (μg/mL), it is necessary to place the sample absorbance into the equation as y.

tional results between the analyte concentration and the device response. The interval be‐ tween the lowest and highest scalar values, which is also called the linearity range, should be determined with both precision and accuracy. The correlation coefficient must be equal to

The calibration curve constructed for tannic acid and tannins and used to quantify *M. ilicifo‐ lia* demonstrated a correlation equation of y = 0.067x + 0.01 and a correlation coefficient of R2

**Figure 5.** Calibration curve constructed using tannic acid concentrations of 1-5 μg/mL at 760 nm used to quantify the

A calibration curve was constructed from rutin to quantify flavonoids in products from *B. forficata* and yielded the correlation equation y = 0.022 + 0.0039 x and correlation coefficient

) shows the ability of the method to provide directly propor‐

in Plant Samples

57

http://dx.doi.org/10.5772/51686

standard operating protocols (SOPs) and the results are presented below.

**4.1. Calibration curves**

The correlation coefficient (R2

tannin content of *Maytenus ilicifolia* (Schrad.) Planch.

or greater than 0.98.

= 0.996 (Figure 5).

R2

= 0.991 (Figure 6).

**Calibration curve:** The calibration curve must be prepared using 50-500 μL aliquots of the coumarin solution, 2 ml of distilled water and 500 μL of the lead acetate solution. The sam‐ ple should be shaken and the final volume should be adjusted to 10 mL with distilled water before transferring 2 mL of this solution to a new test tube and adding 8 mL of hydrochloric acid solution. The final concentration of rutin will be 1-10 μg/mL.

**Measurement procedure:** To quantify the coumarins, 500 μL of the extract should be trans‐ ferred to a test tube. Next, 2 ml of distilled water and 500 μL of lead acetate solution will be added. The sample is shaken and then 7 ml of distilled water are added before transferring 2 mL of this solution to a new test tube and adding 8 mL of hydrochloric acid solution. The samples remain at room temperature for 30 minutes. The spectrophotometer should be ad‐ justed to a 320 nm wavelength and the equipment must be rinsed with distilled water. The test should be performed in at least triplicate and the total coumarin content is expressed as milligrams of coumarin equivalents per gram of the sample extract (mg CE/g).

#### **References:**

Kuster R. A. M., Rocha L. A. M. A. Cumarinas, coronas e cantinas. In: Simões CMO, Schen‐ kel EP, Gosmanm, G, Mello JCP, Mentz LA, Petrovick PR. (ed.) Farmacognosia: da plan‐ ta ao medicamento. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2004, p. 537-556 [34].

Osório O. K., Martins J. L. S. Determinação de cumarina em extrato fluido e tintura de guaco por espectrofotometria derivada de primeira ordem. Brazilian Journal of Pharmaceutical Sci‐ ences 2004; 40 (4) 481-486 [40].

#### **4. Results**

Analysis of the active component levels in raw plant materials and phytomedicines is essen‐ tial for the safety and efficacy of pharmaceutical products [49]. The quantification of active compounds in herbals is still only incidentally performed due to the presence of active phy‐ tocomplexes plants and their extracts [50], which complicates their analysis. Through this framework, the use of standardised extracts focusing on specific groups of active compo‐ nents ensures the chemical homogeneity of the product, which improves product quality [51]. The compounds selected for this quality adjustment process should be the same as the assets in the product [52].

In this way, five products containing *M. ilicifolia* (all plant drugs), four containing *B. forficata* (all plant drugs) and five containing *M. glomerata* (three plant drugs and two fluid extract) sold in pharmacies and health food stores in Recife/PE, Northeast Brazil were obtained. Analysis of the active component concentrations were conducted as listed in the described standard operating protocols (SOPs) and the results are presented below.

#### **4.1. Calibration curves**

into a 50 mL volumetric flask and the final volume should be adjusted with the same sol‐ vent. The final extract concentration will be 10 mg/mL. Liquid products must be evaporated. The dried extract should be dissolved in 80% methanol for a final concentration of 1 mg/mL.

**Calibration curve:** The calibration curve must be prepared using 50-500 μL aliquots of the coumarin solution, 2 ml of distilled water and 500 μL of the lead acetate solution. The sam‐ ple should be shaken and the final volume should be adjusted to 10 mL with distilled water before transferring 2 mL of this solution to a new test tube and adding 8 mL of hydrochloric

**Measurement procedure:** To quantify the coumarins, 500 μL of the extract should be trans‐ ferred to a test tube. Next, 2 ml of distilled water and 500 μL of lead acetate solution will be added. The sample is shaken and then 7 ml of distilled water are added before transferring 2 mL of this solution to a new test tube and adding 8 mL of hydrochloric acid solution. The samples remain at room temperature for 30 minutes. The spectrophotometer should be ad‐ justed to a 320 nm wavelength and the equipment must be rinsed with distilled water. The test should be performed in at least triplicate and the total coumarin content is expressed as

Kuster R. A. M., Rocha L. A. M. A. Cumarinas, coronas e cantinas. In: Simões CMO, Schen‐ kel EP, Gosmanm, G, Mello JCP, Mentz LA, Petrovick PR. (ed.) Farmacognosia: da plan‐ ta ao medicamento. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2004, p.

Osório O. K., Martins J. L. S. Determinação de cumarina em extrato fluido e tintura de guaco por espectrofotometria derivada de primeira ordem. Brazilian Journal of Pharmaceutical Sci‐

Analysis of the active component levels in raw plant materials and phytomedicines is essen‐ tial for the safety and efficacy of pharmaceutical products [49]. The quantification of active compounds in herbals is still only incidentally performed due to the presence of active phy‐ tocomplexes plants and their extracts [50], which complicates their analysis. Through this framework, the use of standardised extracts focusing on specific groups of active compo‐ nents ensures the chemical homogeneity of the product, which improves product quality [51]. The compounds selected for this quality adjustment process should be the same as the

In this way, five products containing *M. ilicifolia* (all plant drugs), four containing *B. forficata* (all plant drugs) and five containing *M. glomerata* (three plant drugs and two fluid extract) sold in pharmacies and health food stores in Recife/PE, Northeast Brazil were obtained.

milligrams of coumarin equivalents per gram of the sample extract (mg CE/g).

acid solution. The final concentration of rutin will be 1-10 μg/mL.

**References:**

56 Latest Research into Quality Control

537-556 [34].

**4. Results**

ences 2004; 40 (4) 481-486 [40].

assets in the product [52].

To quantify the active components, calibration curves with increasing concentrations pro‐ portional to their absorbance were constructed. A correlation equation was obtained from these curves (generally linear) of the type *y = ax + b*, where *y* corresponds to the absorbance of the sample and *x* the concentration. To convert the absorbance values (nm) to sample concentration (μg/mL), it is necessary to place the sample absorbance into the equation as y. The correlation coefficient (R2 ) shows the ability of the method to provide directly propor‐ tional results between the analyte concentration and the device response. The interval be‐ tween the lowest and highest scalar values, which is also called the linearity range, should be determined with both precision and accuracy. The correlation coefficient must be equal to or greater than 0.98.

The calibration curve constructed for tannic acid and tannins and used to quantify *M. ilicifo‐ lia* demonstrated a correlation equation of y = 0.067x + 0.01 and a correlation coefficient of R2 = 0.996 (Figure 5).

**Figure 5.** Calibration curve constructed using tannic acid concentrations of 1-5 μg/mL at 760 nm used to quantify the tannin content of *Maytenus ilicifolia* (Schrad.) Planch.

A calibration curve was constructed from rutin to quantify flavonoids in products from *B. forficata* and yielded the correlation equation y = 0.022 + 0.0039 x and correlation coefficient R2 = 0.991 (Figure 6).

**4.2. Content of active principles**

ins of *M. glomerata* are shown in Table 1.

*Maytenus ilicifolia* Tannins

*Bauhinia forficata* Flavonoids

*Mikania glomerata* Coumarins

Recife/PE, Northeast of Brazil.

statistically different (n = 6, p<0.05).

cient of variation (CV = 37.61%).

The results of quality control of tannins of *M. Ilicifolia*, flavonoids of *B. forficata* and coumar‐

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

**Specie / Marker Sample Concentration ± SD (mg/g) CV (%)**

**Table 1.** Results of quality control of tannins, flavonoids and coumarins contained in products based on *Maytenus ilicifolia (*Schrad.) Planch., *Bauhinia forficata* Link and *Mikania glomerata* Spreng. respectively, sold in pharmacies in

Values are mean ± standard deviation. Values followed by the same letter in column are not

Analysis of variance (ANOVA) is one way to indicate significant differences (p<0.01) for the drugs of *M. ilicifolia*, which is made from five plants. Samples Mi1 (12.57 ± 2.15 mg TAE/g) and Mi5 (11.81 ± 1.0 mg TAE/g) both had higher concentrations of tannins and were not sig‐ nificantly different, whereas at least three of the other samples showed tannins (Figure 8). Comparing the average tannin concentration from different samples showed a low coeffi‐

Analysis of four products containing *B. forficata* showed a significant difference (p<0.01). The sample Bf4 presented the highest flavonoid concentration (65.98 ± 3.62 mg RE/g), whereas samples Bf1 and Bf2 had the lowest concentrations (4.89 ± 0.11 and 7.27 ± 0.39 mg RE/g, re‐ spectively) with a content approximately 13 times lower. These results indicate that there is

no standardisation regarding the flavonoid concentrations in products (Figure 9).

MI1 12.57 ± 2.15a 17.12% MI2 4.04 ± 0.23b 5.75% MI3 5.61 ± 0.55bc 9.76% MI4 7.72 ± 0.84c 10.84% MI5 11.81 ± 1.00a 8.44%

in Plant Samples

59

http://dx.doi.org/10.5772/51686

BF1 4.89 ± 0.11a 2.33% BF2 7.27 ± 0.39a 5.41% BF3 50.38 ± 5.36b 10.64% BF4 65.98 ± 3.62c 5.49%

MG1 3.06 ± 0.20a 6.67% MG2 5.17 ± 0.59b 11.40% MG3 6.80 ± 0.24c 3.46% MG4 1.63 ± 0.20d 12.50% MG5 4.49 ± 0.20b 4.55%

**Figure 6.** Calibration curve constructed from rutin for concentrations of 1-10 μg/mL at 420 nm used to quantify the flavonoids content in *Bauhinia forficata* Link.

The correlation equation and coefficient obtained from the calibration curve used to analyse coumarins in products containing *M. glomerata* were y = 0.049 x + 0.031 and R2 = 0.994 (Figure 7), respectively.

**Figure 7.** The calibration curve constructed using 1,2-benzopyrone in concentrations of 1-12 μg/mL using 320 nm ex‐ citation to quantify the coumarin content in *Mikania glomerata* Spreng.

#### **4.2. Content of active principles**

**Figure 6.** Calibration curve constructed from rutin for concentrations of 1-10 μg/mL at 420 nm used to quantify the

The correlation equation and coefficient obtained from the calibration curve used to analyse

**Figure 7.** The calibration curve constructed using 1,2-benzopyrone in concentrations of 1-12 μg/mL using 320 nm ex‐

citation to quantify the coumarin content in *Mikania glomerata* Spreng.

= 0.994 (Figure

coumarins in products containing *M. glomerata* were y = 0.049 x + 0.031 and R2

flavonoids content in *Bauhinia forficata* Link.

58 Latest Research into Quality Control

7), respectively.

The results of quality control of tannins of *M. Ilicifolia*, flavonoids of *B. forficata* and coumar‐ ins of *M. glomerata* are shown in Table 1.

**Table 1.** Results of quality control of tannins, flavonoids and coumarins contained in products based on *Maytenus ilicifolia (*Schrad.) Planch., *Bauhinia forficata* Link and *Mikania glomerata* Spreng. respectively, sold in pharmacies in Recife/PE, Northeast of Brazil.

Values are mean ± standard deviation. Values followed by the same letter in column are not statistically different (n = 6, p<0.05).

Analysis of variance (ANOVA) is one way to indicate significant differences (p<0.01) for the drugs of *M. ilicifolia*, which is made from five plants. Samples Mi1 (12.57 ± 2.15 mg TAE/g) and Mi5 (11.81 ± 1.0 mg TAE/g) both had higher concentrations of tannins and were not sig‐ nificantly different, whereas at least three of the other samples showed tannins (Figure 8). Comparing the average tannin concentration from different samples showed a low coeffi‐ cient of variation (CV = 37.61%).

Analysis of four products containing *B. forficata* showed a significant difference (p<0.01). The sample Bf4 presented the highest flavonoid concentration (65.98 ± 3.62 mg RE/g), whereas samples Bf1 and Bf2 had the lowest concentrations (4.89 ± 0.11 and 7.27 ± 0.39 mg RE/g, re‐ spectively) with a content approximately 13 times lower. These results indicate that there is no standardisation regarding the flavonoid concentrations in products (Figure 9).

sample Mg4 had the lowest level (1.63 ± 0.20 mg CE/g), with an approximately four times

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

in Plant Samples

61

http://dx.doi.org/10.5772/51686

**Figure 10.** Concentration of coumarins (mg CE/g) contained in products from *Mikania glomerata* Spreng. sold in Re‐

The quantitative analysis of raw vegetables and phytomedicines is a fundamental quality control process that leads to security, stability, consistency and effectiveness in the produced phytomedicines [49]. It is important to emphasise the need for standardisation in analysing herbal medicines to determine the concentration of their active components in raw vegetable

This chapter provides easily reproducible standard operating procedures (SOPs) for the quality control of raw materials and herbal plants to ensure a minimal standard of quality in products sold. The implementation of these SOPs allows for the analysis of samples sold in establishments in Recife/PE and reveals an inconsistency in the concentration of tannins, fla‐

The low level of these metabolites may alter their effectiveness and more rigorous quality control and standardisation of these products is required to prevent compromising their

lower coumarin concentration.

cife/PE, Brazil.

**5. Conclusion**

therapeutic activity.

materials as well as for species identification.

vonoids and coumarins within these products.

**Figure 8.** Concentration of tannin (mg TAE/g) contained in products from *Maytenus ilicifolia* (Schrad.) Planch. Sold in Recife/PE, Brazil.

**Figure 9.** Concentration of flavonoids (mg RE/g) contained within *Bauhinia forficata* link. sold in Recife/PE, Brazil.

Of the five products from *M. glomerata*, three were plant drugs and two were fluid extracts. The plant drugs were extracted with ethanol as recommended by the Brazilian Pharmaco‐ poeia 4th Edition to remove discrepancies from the results [53]. Both the extracts and liquids were evaporated to dryness. The one way ANOVA showed significant differences between the products (p<0.01) and sample Mg3 had the highest level (6.80 ± 0.24 mg CE/g), whereas sample Mg4 had the lowest level (1.63 ± 0.20 mg CE/g), with an approximately four times lower coumarin concentration.

**Figure 10.** Concentration of coumarins (mg CE/g) contained in products from *Mikania glomerata* Spreng. sold in Re‐ cife/PE, Brazil.

The quantitative analysis of raw vegetables and phytomedicines is a fundamental quality control process that leads to security, stability, consistency and effectiveness in the produced phytomedicines [49]. It is important to emphasise the need for standardisation in analysing herbal medicines to determine the concentration of their active components in raw vegetable materials as well as for species identification.

#### **5. Conclusion**

**Figure 8.** Concentration of tannin (mg TAE/g) contained in products from *Maytenus ilicifolia* (Schrad.) Planch. Sold in

**Figure 9.** Concentration of flavonoids (mg RE/g) contained within *Bauhinia forficata* link. sold in Recife/PE, Brazil.

Of the five products from *M. glomerata*, three were plant drugs and two were fluid extracts. The plant drugs were extracted with ethanol as recommended by the Brazilian Pharmaco‐ poeia 4th Edition to remove discrepancies from the results [53]. Both the extracts and liquids were evaporated to dryness. The one way ANOVA showed significant differences between the products (p<0.01) and sample Mg3 had the highest level (6.80 ± 0.24 mg CE/g), whereas

Recife/PE, Brazil.

60 Latest Research into Quality Control

This chapter provides easily reproducible standard operating procedures (SOPs) for the quality control of raw materials and herbal plants to ensure a minimal standard of quality in products sold. The implementation of these SOPs allows for the analysis of samples sold in establishments in Recife/PE and reveals an inconsistency in the concentration of tannins, fla‐ vonoids and coumarins within these products.

The low level of these metabolites may alter their effectiveness and more rigorous quality control and standardisation of these products is required to prevent compromising their therapeutic activity.

#### **Author details**

Elba Lúcia Cavalcanti de Amorim1\*, Valérium Thijan Nobre de Almeida de Castro1 , Joabe Gomes de Melo2 , Allan Jonathan Chernichiarro Corrêa1 and Tadeu José da Silva Peixoto Sobrinho3

[9] Lewis, N. G., & Yamamoto, E. (1989). Tannins: their place in plant metabolism. In: Hemingway RW, Karchesy JJ. (Ed) Chemistry and significance of condensed tannins.

Standard Operating Procedures (SOP) for the Spectrophotometric Determination of Phenolic Compounds Contained

in Plant Samples

63

http://dx.doi.org/10.5772/51686

[10] Heil, M., Baumann, B., Andary, C., Linsenmair, K. E., & Mckey, D. (2002). Extraction and quantification of "condensed tannins" as a measure of plant anti-herbivore de‐

[11] Bruneton, J. (1991). Elementos de Fitoquimica y Farmacognosia. 1ª ed. Zaragoza: Ed.

[12] Harborne, J. B., Palo, R. T., & Robbins, C. T. (1991). Plant defenses against mammali‐

[13] Gobbo-Neto, L., & Lopes, N. P. (1991). Plantas medicinais: fatores de influência no

[14] Verpoorte, R. (1998). Exploration of nature's chemodiversity: the role of secondary metabolites as leads in drug development. *Drug Development Trends*, 3(5), 232-238.

[15] Wills, R. B. H., Bone, K., & Morgan, M. (2000). Herbal products: active constituents

[16] Kilkuskie, R. E., Kashiwada, Y., Nonaka, G., Nishioka, I., Bodner, A., Cheng, Y., & Lee, K. (1992). HIV and reverse transcriptase inhibition by tannins. *Bioorganic & Me‐*

[17] Chung, K., Wei, C., & Johnson, M. G. (1998). Are tannins a double-edged sword in

[18] Ferreira, P. M., Oliveira, C. N., Oliveira, A. B., Lopes, M. J., Alzamora, F., & Vieira, M. A. R. (2004). A lyophilized aqueous extract of Maytenus ilicifolia leaves inhibits his‐ tamine-mediated acid secretion in isolated frog gastric mucosa. *Planta*, 219(2),

[19] Ming, L. C., Castro, D. M., & Delachiave, M. E. (1998). Plantas medicinais aromáticas

[20] Pereira, A. M. S., Rodrigues, D. C., Cerdeira, R. M., & França, S. C. (1993). Isolamento de metabólitos de maytenus associadas à ação anti-úlceragástrica. 12º. Simpósio de

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\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Pharmacy, Federal University Pernambuco, Recife/PE, Brazil

2 Department of Biology, Federal Rural University of Pernambuco, Brazil

3 School of Medicine, Federal University of Tocantins, Palmas/TO, Brazil

#### **References**

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**Author details**

**References**

Joabe Gomes de Melo2

62 Latest Research into Quality Control

Tadeu José da Silva Peixoto Sobrinho3

\*Address all correspondence to<EMAIL_ADDRESS>
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do Rio Grande do Sul;, 403-434.

court Brace and Company , 318p.

Elba Lúcia Cavalcanti de Amorim1\*, Valérium Thijan Nobre de Almeida de Castro1

1 Department of Pharmacy, Federal University Pernambuco, Recife/PE, Brazil

2 Department of Biology, Federal Rural University of Pernambuco, Brazil

3 School of Medicine, Federal University of Tocantins, Palmas/TO, Brazil

, Allan Jonathan Chernichiarro Corrêa1

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**Chapter 4**

**Microbial Quality of Medicinal Plant Materials**

The use of medicinal plants is continually expanding worldwide. The increasing search for therapeutic agents derived from plant species is justified by the emergence of diseases, yet without proper treatment, and the growth of scientific knowledge about the herbal medi‐ cines as important treatment alternatives. Therefore, the quality and safety of herbal prepa‐ rations are also of great concern [1]. The reference [2] explained that quality is the basis of reproducible efficacy and safety of herbal drugs, and to ensure the standard of research on herbal medicines, the quality of the plant materials or preparations is of utmost importance. With the ever increasing use of herbal medicines and the global expansion of the herbal medicines market, safety has become a concern for both health authorities and the public in many countries. This is because many contaminants and residues that may cause harm to

The microbial load of plants is the result of a series of influences (Figure 1). By their origin, herbal drugs are subject to contamination by microorganisms from soil, air and water may be present potentially pathogenic microorganisms to man. Microbial contamination of me‐ dicinal herbal can be influenced by environmental factors such as temperature, humidity and extent of rainfall during pre-harvesting and post-harvesting periods, handling practices and the storage conditions of crude and processed medicinal-plant materials. In order to im‐ prove the purity and safety of the products, observation of basic hygiene during prepara‐ tion, standardization of some physical characteristic such as moisture content, pH and

The presence of microbial contaminant in non sterile pharmaceutical products can reduce or even inactivate the therapeutic activity of the products and has the potential to adversely af‐ fect patients taking the medicines. As herbal medicinal products are complex mixtures

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