Malihe Tabarrai,Mozhgan Mehriardestani,Sharareh Hekmat,Fatemeh Nejatbakhsh,Fatemeh Moradi,＊

1Department of Traditional Medicine,School of Persian Medicine,Tehran University of Medical Sciences,Tehran,Iran.

2Department of Traditional Pharmacy,School of Persian Medicine,Tehran University of Medical Sciences,Tehran,Iran.

Abstract

Keywords:Vitis vinifera L.,Grape,Oxidation,Immunomodulation,Inflammation,Abortion,Persian medicine

Background

Threatened abortion in International Classification of Diseases 10threvision (2019) is defined: “Uterine bleeding from a gestation of fewer than 20 weeks without any cervical dilatation.It is characterized by vaginal bleeding,lower back discomfort,or midline pelvic cramping and a risk factor for miscarriage” [1].Threatened miscarriage is a prevalent disorder and almost a quarter of pregnant women are diagnosed with significant vaginal bleeding in early pregnancy.Finally,about 50% of these pregnancies lead to abortion [2].The medications including progesterone,hCG (human chorionic gonadotropin) or uterine muscle relaxant are prescribed empirically for threatened abortion.Although some of these medications have important physiological roles in pregnancy development,the clinical data are insufficient to advise them [3].In this regard,medicinal herbs could help us to discover new treatments [4].There are fewin vivoorin vitrostudies on medicinal herbs for miscarriage prevention.While throughout the world,many pregnant women use medicinal herbs to prevent threatened abortion [5-9].One of the medicinal herbs that can have functions on the immune and endocrine factors involved in threatened abortion is grape (Vitis vinifera L.).There are several phenolic compounds,flavonoids,and stilbenes in different grape components including seed,fruit,skin,pomace,and stem [10].For example,resveratrol,a polyphenol found only in grapes and not available in other vegetables and fruits,was shown to exert an antioxidant activity on some prenatal metabolic disorders like low protein diet,maternal obesity,gestational diabetes mellitus and hypoxia [11,12].In addition to antioxidant properties,many investigations have been conducted on the grape pharmacological activities such as anticancer,anti-inflammatory,antibacterial and antidiabetic effects with skin,liver,heart and neural system protection[10].

From the viewpoint of the Persian medicine,pregnancy loss has three main causes,including trauma,maternal factors,and fetal factors[13,14].The discontinuation of uterine bleeding is crucial in early pregnancy,because even if it does not lead to abortion,it causes weakness in fetus and variety of mild-to-severe illnesses in newborn after birth [15].Therefore,Persian physicians controlled bleeding during pregnancy and administrated tonic foods and“fetus protection” medicines to strengthen mother and fetus.Undeniably,main cause of abortion cannot be resolved during pregnancy in most cases,because most treatments is not safe in pregnant woman.So,the main treatment for this condition is after miscarriage and before proceeding to the next pregnancy.In Persian medicine references such asThe Canonof Medicine(written by Avicenna,980 A.D.-1037 A.D.)andGreat Elixir(written by Mohammad Azam Khan,1814 A.D.-1902 A.D.),there were many medications and foodstuffs for reduce bleeding in early pregnancy[13,16],and the grape was one of the remedies for fetal protection [17,18].This hypothesis discusses the possible role of grape in fetus protection and abortion prevention.

The hypothesis

It is hypothesized that grape can prevent or control threatened abortion with its immunomodulatory,anti-inflammatory and antioxidant activities and inhibiting uterine contractions.

Evaluation of the hypothesis

Grape in Persian medicine

Grape has been namedEnabin Persian medicine.Grape is one of the best fruits,because it has high nutrition and is a body booster.Grape and grape molasses also produces good bloodhumor(Humorsare basic substances and fluids found in human body,including black bile,yellow bile,phlegm,and blood.)[17].Grapevine calledKarmin Persian medicine texts.In the traditional Persian medicine references asMakhzan al-Advieh(written by Aghili,1670 A.D.-1749 A.D.) andTohfat al-Momenin(written by Mohammad Momen,16th century),it was mentioned that its leaf extract could prevent abortion.Unripe grape reduces laxity of tissues and strengthen the body.The paste of unripe grape,in addition to protecting the fetus,fortifies the viscera and decreases inflammation of stomach [17,18].Rhazes,a great scientist of the 9th century A.D.,inAl-Hawi(written by Rhazes,854 A.D.-925 A.D.) noted that verjuice(unripe grapes juice,used as vinegar) could fortify the stomach of pregnant women and prevent the fetus from abortion[19].

Mechanisms of threatened abortion

Although inflammation has a necessary role in successful implantation and healthy development of pregnancy,excessive inflammation and immune suppression are the crucial mechanisms of miscarriage.Also,inflammation has more adverse pregnancy outcomes like preeclampsia[20,21].

Balance of Th (T helper) 1/Th2 to shift toward Th2 is essential for maintaining a healthy gestation[20,21].Th2 cells are involved in humoral immunity.Th2 cytokines [IL (interleukin)-4,IL-5,IL-6,IL-9,and IL-10] cause fetal immune protection.On the other side,Th1 cells produce IL-2,IFN-γ (interferon-γ),and TNF-α (tumor necrosis factor-α) and have an essential role in autoimmune diseases and fetal immune impairment.So,the primary factor causing abortion is strengthening of Th1 cell-mediated immunity[21,22].

Progesterone also can enhance Th2 immune reaction and attenuate Th1 immune reaction.It seems progesterone is a more important factor in the prevention and treatment of miscarriage because progesterone modulates the maternal immune system and suppresses the inflammatory response.It also develops a uterus,nourishes eggs,improves uteroplacental circulation,supports the luteal phase and decreases contraction of uterus smooth muscle after implantation.So,insufficient secretion of progesterone plays a vital role in threatened abortion and recurrent miscarriage[22-24].

Estrogens also are needed for maintenance and continuance of pregnancy [25].They could inhibit the immune response of the mother to the fetus and so prevent miscarriage [26].Estrogens increase expression of the progesterone and strengthen the progesterone activities [27].Furthermore,hCG from trophoblastic cells decrease the immune activity of lymphocytes and protect the fetus from maternal immune attack [22].Some disorders like hypercoagulable state also result in abortion through immunological rejection or placental damage[3].

Oxidative stress or toxic agent are incriminated for initiation of spontaneous abortion.Therefore,antioxidants with detoxifying and repairing oxidation damaged can be useful in miscarriage[21,28,29].

NO (nitric oxide) and PGs (prostaglandins) have different effects on abortion and pregnancy periods and the mechanisms of action are not precisely clear.PGs and NO donors are administrated for cervical ripening before first-trimester surgical termination of pregnancy[30,31].In preterm labor and also during cervical ripening in late pregnancy,NO is increased in cervical fluid[32].

However,in early pregnancy,NO is required for embryonic development.On the other side,Serum NO concentration in threatened abortion looks like normal pregnancy [31].However,elevation in serum level of NO in recurrent abortion [33] and decreased NO in missed abortion [31] have been detected.PGs are also needed for successful embryonic implantation.They can suppress rejection of the fetal allograft[34].Use of nonsteroidal anti-inflammatory drugs around the time of conception leads to abnormal implantation and miscarriage [3,35].However,on the other side,inadequate inhibition of uterine PGs may be correlated with abortion[30].

In addition to the above mentioned,more factors are involved in abortion mechanisms such as:activation of cytotoxic T cells,natural killer cells [20-22,36];elevation of Th17 and related cytokines like IL-17,IL-23 [21,36],ICAM-1 (intracellular adhesion molecules-1),VCAM-1 (vascular cell adhesion molecule-1),IL-2 [22,37],T-cell immunoglobulin mucin-3 and adipsin [36];decreased activation of regulatory T cells [21,36],TGF-β,[37,38];premature or aberrant activation of NF-κB[21],etc.

Putative mechanisms of grape to prevent threatened abortion

Grape polyphenols have many biological activities that could prevent and treat several diseases.The most critical polyphenolic compounds in grape are flavonoids,flavanols,anthocyanins,resveratrol,quercetin,epicatechin,etc.[39,40].Polyphenolic compounds of grape,like resveratrol,have been shown to exhibit immunomodulatory,anti-inflammatory and antioxidant effects and also inhibit uterine contractions[11,41-43].These properties during pregnancy could prevent or control threatened miscarriage(Figure 1).

Figure1 Expected mechanisms of grape to prevent threatened abortion

Immunomodulatory and anti-inflammatory activities.The elevation of anti-inflammatory cytokine(such as IL-4 and IL-10) and reduction of proinflammatory cytokines (like IL-1β,IL-6,IL-8,IL-17,TNF-α and IFN-γ) are involved in preventing abortion [21].Elevation of TNF-α,and IL-6 had been seen respectively to cause spontaneous and recurrent abortion [21,37].On the other hand,decreased TNF-α and IFN-γ and increased IL-4 and IL-10 levels could inhibit the fetal allograft[22,37].

Grape and its related compounds have complex functions in anti-inflammatory properties.Raisin (both fruit and seed) inhibits IL-8 secretion through various mechanisms [44].Grape juice inhibits expression of TNF [45].Grape seed and grape marc reduce IL-1β,IL-8,and TNF-α [46].Grape skin or flesh inhibit TNF-α and IFN-γ and enhance significantly IL-4 [11].Grape powder extract reduces IL-1β,IL-6,IL-8,and TNF-α.Grape-seed procyanidin extract reduces IL-6 and TNF-α and elevates anti-inflammatory cytokines[47].Grape-seed epicatechin inhibits IL-1β and IL-6 production [48].Resveratrol decreases the levels of IL-1β[42].

In inflammation,alongside pro-inflammatory cytokines,other mediators such as NO and PGs elevate[49].Reduction of PGE2 (prostaglandin E2) and NO due to reduction of COX-2 (cyclooxygenase-2) and iNOS (inducible NO synthase) were observed in threatened abortion treatment [21].Piceatannol,a grape polyphenol,acts to prevent and treat inflammatory bowel diseases with reducing of pro-inflammatory cytokines,NO and PGE2[49].Other grape compounds including resveratrol,grape seed,and grape juice inhibit expression of COX-2 and iNOS and decrease prostaglandin D2 level [42,45,47,50,51].

Increasing expression of peroxisome proliferator-activated receptor-γ is the other mechanism that could block inflammation and decrease oxidative stress.Grape powder extract and anthocyanins may be a role in miscarriage prevention or treatment with this mechanism[36,47].

Furthermore,NF-κB plays a role in proinflammatory genes expression.Inhibition of NF-κB is significant in the treatment of several diseases [21].Several products from grape decrease NF-κB activation[45-47,52,53].

Reduction of expression of ICAM-1 and VCAM-1 also was seen in the treatment of threatened abortion[21].Activin,a proanthocyanidin in grape seed extract,attenuates the levels of ICAM-1 and VCAM-1 significantly.Raisins [54],red wine,grape juice [55]and quercetin [56] decrease only the ICAM-1 expression.It seems that grape products reduce TNF-α expression and then increase ICAM-1 expression [55]via inhibition of NF-κB pathway[46,47,55].

In addition,in subchorionic hemorrhage,the increase of EGF (epidermal growth factor) and PDGF(platelet-derived growth factor) was seen [21].Some products of grape inhibit the levels of EGF and PDGF[47,50,57,58].

Anti-oxidant activities.Decreased antioxidant level in pregnant body is thought to play a role in the etiology of abortion [59],and some dietary supplements have functions in preventing miscarriage with antioxidant and anti-inflammatory activities [21,60].Even an increased intake of antioxidants by men with sperm disorders,can improve gestational outcomes in couples with history of recurrent abortions[61].Many research suggest that grape seed has many health-promoting benefits because of its antioxidant effects.The antioxidant power of grape seed proanthocyanidins is 50 times greater than that of vitamin C and 20 times greater than that of vitamin E [39].Also,antioxidant activity of grape molasses is more than grape and raisins[62].The high melatonin content in the seeds of edible plants like grape,could protect fetus from oxidative stress [63].In pregnancy,red grapes seeds extract shows prophylactic effects against oxidative stress [29].Flavonoids can protect DNA from oxidation,especially in inflamed tissues[64].So,when a pregnant woman uses flavonoid in her diet,like grape quercetin,she protects her fetus from reproductive toxicants[65].

Anti-contraction.Uterine contractions are induced by several factors such as PGF2α,oxytocin,acetylcholine,carbachol,calcium channel activator,etc.Grape resveratrol inhibits uterine contractions induced with these mechanisms and could imitate calcium channel blockers in the plasma membrane[66].

Hormonal activities.Progesterone acts with hormonal and immunological mechanisms to maintain pregnancy[23].Grape leaves could affect progesterone [67,68].Although,estrogens are essential in the maintenance of pregnancy [25],in the other,elevated estrogen is linked to unexplained recurrent miscarriage [69,70].Grape antioxidants have both estrogenic and antiestrogenic properties.Because the structure of some grape antioxidants,like resveratrol,quercetin,and catechin,are similar to the steroid hormones[50].Anti-stress activities.Biological stress can increase TNF-α and decrease TGF-β2 [38].Decreased TNF-α and increased TGF-β levels can also inhibit fetal rejection [37].So,the grape products which decreased TNF-α probably had a role in stress-triggered abortion prevention [11,45-47].On the other,CD8+ T cells immunization could prevent the stress-triggered abortion [38] and proanthocyanidins of grape seed could stimulate CD8+effector T cells[71].

Anti-candida activities.Although vaginal candidiasis during pregnancy does not influence abortion,treatment of candidiasis could prevent preterm labor[41].Resveratrol has fungicide effects[43].Grape seed extracts could inhibit Candida infections because of the high level of flavanols.Epigallocatechin gallate,a grape flavanol,has effect on the several fungi,including Candida albicans[72].

Consequences of the hypothesis and discussion

This proposed hypothesis offers grape may have salutary effects on threatened abortion in pregnant women or women who are going to become pregnant.If the hypothesis is right,the grape can prevent threatened abortion or reduce miscarriage rate with different mechanisms and can be recommended as a natural fetal protective product.Even so,it still suggests that more investigations should be done to identify grape’s various mechanisms involved in abortion around and during pregnancy.

In addition to numerous useful properties of grape in fetal protection,some findings do not support this hypothesis at first glance.As mentioned above,in the prevention of abortion,Th2 anti-inflammatory cytokines like IL-4 and IL-10 increased and pro-inflammatory Th1 cytokines such as IFN-γ decreased [21,22].However,in two studies,grape seed products increased IFN-γ (Th1 cytokine) and suppressed the levels of IL-10 and IL-4[71,73].These deleterious grape seed effects can be justified by several ways.Firstly,excessive inflammation is one of the causes of abortion.Whereas,inflammation has a pivotal role in successful embryonic implantation [21].Secondly,in pregnancy,the relative changes of Th1 and Th2 cytokines are more important than the absolute concentration changes between Th1 or Th2 productions [22].Finally,although,there were higher ratio of Th1/Th2 in recurrent miscarriage [74] and higher Th1 and lower Th2 in threatened abortion [75],in Kalinka study,no relation to Th1 and Th2 concentrations was found in threatened abortion [76].So,maybe grape seed does not cause abortion.

As pointed out earlier,iNOS/NO or COX2/PGs worked like a double-edged sword in pregnancy.iNOS has accumulated in many autoimmune and chronic inflammatory disorders like SLE (systemic lupus erythematosus),but the exact function of iNOS is not clear.For example,SLE could cause abortion with immunological rejection or placental damage.In some diseases,iNOS is elevated with proinflammatory Th1 reactions for limitation of bacterial invasion,local immune reactions and so tissue destruction against Th1 responses [3,77].Maybe it can be said,controlling excessive inflammation is also crucial in NO responses.Moreover,grape decreases excessive inflammation with several mechanisms as discussed in this article.In the other words,hyperactivation of iNOS/NO or COX2/PGs pathways can lead to abortion,not only activation of them.

Conclusion

Based on the described mechanisms of action in above,we can anticipate that grape protects fetus.Of course,further clinical investigations are necessary.According to Rhazes’ (865 A.D.-925 A.D.) opinion about the priority of food therapy to drug therapy [78],we suggest the design of a study with edible grape products.In this regard,we recommend to evaluate nutritional and therapeutic effect of grape foodstuff in maternal diet in clinical study,such as grape seed oil,grape juice or grape molasses.In the next steps,studies can be designed with grape leaves extract in threatened abortion.