1. Introduction {#s0005}
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Chronic and degenerative diseases in middle-aged women such as cancer, cardiovascular, and metabolic diseases are increasing. These diseases are the leading cause of death and disability disorders in developing countries. [@b0075] showed that large changes in behavior patterns due to the decreasing consumption of vegetables and fruits, which plays an important role in increasing the incidence of chronic and degenerative diseases. Bioactive compounds in fruits and vegetables such as polyphenols, flavonoids, anthocyanins, micronutrients, minerals and vitamins, have antioxidant and anti-inflammatory activities ([@b0140]). Therefore, they possess preventive and therapeutic potencies against diseases ([@b0010], [@b0160]).

Date palm (*Phoenix dactilyfera* L.) seed is one of the rich source of polyphenols and flavonoids ([@b0035]). It has been extensively investigated for pharmacological activities such as anti-inflammatory ([@b0115]), immuno-stimulant ([@b0105]), antidiabetic ([@b0045]), antibacterial ([@b0025]), antiviral ([@b0060]), and antioxidant ([@b0020], [@b0035], [@b0050], [@b0090]). In previous studies, the date palm seeds were proven to work as anti-inflammatory ([@b0120], [@b0125], [@b0120]), and antiatherogenic food substance ([@b0110]). Recent study showed that various compounds act as anti-inflammatory by metabolomic approach ([@b0005]). Anti-inflammatory effect of date seeds in human has not been adequately studied, especially in middle-aged women. This study, therefore, aims to examine the anti-inflammatory mechanisms of steeped date palm seed in middle-aged women.

2. Material and methods {#s0010}
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2.1. Study design and participants {#s0015}
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This is a quasi-experimental design with a pre and post-test approach. Data were obtained from a total of 30 randomly selected middle-aged women living at Gununglurah, Cilongok District, Banyumas. The inclusion criteria were women between the age of 45--60 years, with no history of metabolic disease by measuring their blood glucose and total cholesterol levels. These women live in farming community and perform the domestic tasks of housewives. In this study, each subject received a single dose of 2.5 g seed powder per day for 14 days. The seed powder was consumed using 250 mL of boiling water. Subjects signed an informed consent before participating in this study, which was conducted after obtaining approval of ethical clearance from the medical research ethics committee of the Faculty of Medicine University of Negeri Sebelas Maret Surakarta, number: 541/IV/HREC/2019.

2.2. Preparation of date seeds powder {#s0020}
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Deglet Nour dates collected from Tunisia, were obtained from a market at Purwokerto, Banyumas Regency, Indonesia. Seeds were manually separated from the date flesh, cleaned with water, selected, and dried for one day in sunlight. The dried seeds were roasted at medium temperature, crushed in a blender and filtered to obtain a fine powder.

2.3. Measurement pro-inflammatory mediators {#s0025}
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A 3 mL blood sample was taken through the median cubital vein before and after treatment. The level of IL-1β, TNF-α, IL-6, IL-12, TGF-β, COX-1, COX-2, and prostaglandin E2 were examined by ELISA kit (BT Laboratories, Shanghai) based on the manufacturer\'s protocol, using an ELISA machine Reader (Labotrone, Germany).

2.4. Statistical analysis {#s0030}
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The mean score was compared before and after treatment with all data presented as mean ± SEM. Differences in scores before and after treatment were analyzed using the student *t*-test. The graph pad software (GraphPad Prism, San Diego, CA) was used to analyze statistical and graphical data, with a p value \< 0.05 considered significant.

3. Results {#s0035}
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3.1. Characteristics of participants {#s0040}
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Thirty subjects were initially recruited, however, eight were unable to complete the treatment. Therefore, a total of 22 subjects were included in the statistical analysis. The age of subjects ranged was from 51 to 57 years, with a healthy weight status and an average body mass index of 24 kg/m^2^, blood glucose below 140 mg/dL, and total cholesterol below 200 mg/dL ([Table 1](#t0005){ref-type="table"}).Table 1Respondent characteristic.VariableRangesMeanAge (years)51-5755Blood pressure Systole (mmHg)100-175135 Diastole (mmHg)65-10093Body mass index (kg/m^2^)23.5--24.524

3.2. Expression of proinflammatory cytokines {#s0045}
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The expression of IL-1β and TGF-β reduced significantly after the consumption of date palm seeds in the post-treatment stage, while TNF-α, IL-12, and IL-6 did had no significant change ([Fig. 1](#f0005){ref-type="fig"}). This result suggests the date palm seed reduces IL-1β and TGF-β produced in healthy middle-aged women.Fig. 1**Treatment date palm seed decreases IL-1β and TGF-β expression.** The cytokines levels were measured at day 0 (pre-treatment) and day 15 (post-treatment) by enzyme-linked immunosorbent assay (ELISA). Mean ± SE are presented (n = 22). Asterisks indicate student *t*-test significance values; \*\*P \< 0.01, \*P \< 0.05. ns = not significant.

3.3. The role of dates palm seed on cyclooxygenase pathway {#s0050}
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The cyclooxygenase (COX) pathway is important in arachidonic acid metabolism related to the inflammatory process ([@b0080]). Therefore, the expression of COX-1, COX-2, as well as prostaglandin E2 level, showed that these mediators are important markers on the COX pathway. A significant decrease in the level of COX-1, COX-2 and PGE2 was observed in the post-treatment stage compared with pre-treatment of steeped of date palm seeds ([Fig. 2](#f0010){ref-type="fig"}). Interestingly, the expression of COX-2 substantially decreased (p \< 0.001), which indicates that the inducible expression of COX-2 in middle-aged women was decreased by consumption of date palm seeds.Fig. 2**Treatment date palm seed decreases COX-1, COX-2, PGE2 level.** The enzyme/mediator levels were measured at day 0 (pre-treatment) and day 15 (post-treatment) by enzyme-linked immunosorbent assay (ELISA). Mean ± SE are shown (n = 22). Asterisks indicate student *t*-test significance values; \*\*P \< 0.01, \*\*\*\*P \< 0.001.

4. Discussion {#s0055}
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Several studies have been conducted to examine the potential of date palm seeds as an anti-inflammatory ([@b0015]) *in vitro* and *in vivo* ([@b0035], [@b0085], [@b0115]). However, no anti-inflammatory activity studies have been conducted on date palm seeds in humans, women middle age. This study, found that the expression of IL-1β, TGF-β, COX-1 and COX-2 decreased after the administration of date palm seeds to middle age women.

Studies showed that the elderly and middle age women experience an increase in free radicals due to various factors such as aging, food, pollution, and excess activity. Free radicals in the body causes oxidation in normal cells, thereby, leading to inflammation and diseases ([@b0135], [@b0145]). Lymphocytes and other immune cells produce cytokines such as IL-1β and TGF-β when there are inflammatory stimuli.

Interleukin-1β is highly elevated in chronic diseases such as obesity, osteoarthritis, and gout ([@b0030]). IL-1β affects lymphocytes and macrophages, induces the formation of prostaglandins, colony stimulating factors and other cytokines ([@b0165]). IL-1β expression is stimulated by various stimuli including sterile stimulus through the formation of inflammasomes ([@b0130]). Since middle age women have developed accumulated agents capable of activating the inflammasome formation, such as cholesterol and uric acid ([@b0095]), it is therefore, important to determine the ability of the date palm seed to reduce the expression of IL-1β. Another interesting aspect is to reveal the role of date palm seeds in inhibiting IL-1β expression, which is further investigated in future.

Although TGF-β was initially called as anti-inflammatory cytokines, it also induces inflammatory Th17 cells differentiation ([@b0170]). Although the role TGF-β in inflammation remains unclear, the date palm seed showed its ability to decrease.

Cyclooxygenase pathway has been well-established for their role in inflammation ([@b0055]). Conversion of phospholipids to arachidonic acid is mediated by the enzyme cyclooxygenase. COX-1 is constitutively expressed and plays a role in the protection of the gastric mucosa. COX-2 is expressed inducibly by an inflammatory stimulus ([@b0155]). Furthermore, various stimuli tend to induce COX-2 expression, which tends to occur with increasing age. In this study, COX-2 expression greatly decreased after the administration of date palm seeds to middle age women. Both enzymes induce the conversion of arachidonic acid into prostaglandins, e.g. PGE2 which plays a role in the vasodilation of blood vessels and increased vascular endothelial permeability ([@b0070]). The expression of both COX enzymes is inhibited by date palm seeds, with a decrease in PGE2 production.

This study is limited to high of dropping out because the subject did not complete the consumption of date palm seed powder as assigned. In addition, the food consumed by the subjects and likely to affect the level of proinflammatory mediators in the human body were not recorded.

Date palm seeds may work as an anti-inflammatory and improve the performance of the immune system ([@b0105]). [@b0100] stated that it works to suppress NF-κB, COX-1 and COX-2 enzymes, thereby, decreasing pro-inflammatory mediators. The anti-inflammatory activity of the aqueous extract of date palm seeds is related to components of polyphenols such as caffeoyl hexoside, 5-O-caffeoyl shikimic acid isomers, hydrocaffeic acid, and isorhamnetin ([@b0150], [@b0065]). This ingredient has also been proven safe for liver and kidney ([@b0040]), therefore, it can be consumed regularly by middle age women to maintain health status, improve immune systems, and prevent chronic diseases. Clinical implications obtained from this study shows that the physicians may suggest the steeped of palm seed powder as a functional beverage.

5. Conclusion {#s0060}
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Dates seeds can act as an anti-inflammatory by reducing interleukin-1β, TGF-β, cyclooxygenase-1 and -2 expression. Regular consumption of date palm seeds increases the body\'s immunity and prevent chronic diseases.

Declaration of Competing Interest
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The authors declared that there is no conflict of interest.

 {#s0080}

**Availability of Data and Materials**

All data generated or analyzed during this study are included in this manuscript. Raw data are available from the corresponding author on a reasonable request.

Acknowledgments {#s0065}
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The authors are grateful to Dr. Hernayanti for her laboratory support; to Lita Kusuma and Nina Setiowati for critically reviewing the manuscript.

Funding {#s0070}
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This study was supported by The Ministry of Research Technology and Higher Education of Indonesia through PDUPT funding.

Authors' contributions {#s0075}
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SY, W, AI conceived and designed the experiments; SY, W, AI performed the experiments; SM analyzed the data; SY, W, AI contributed chemicals/reagents/materials/analysis tools; SY and SM wrote the paper. The authors read and approved the final manuscript.

Peer review under responsibility of King Saud University.
