Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preliminary Investigation for Anticancer Activity

Introduction: Attention related to scientifi c and research interest towards naturally derived compounds from medicinal plant products as they are considered to have less toxic side effects as compared to current treatments such as chemotherapy. Medicinal plant produces naturally occurring secondary metabolites especially fl avonoids which are being investigated for their anticancer activities leading to the development of new clinical drugs. ObjectiveIn this regard, immunological studies were conducted to investigate its immunosuppressive and cytotoxic activity of fl avonoids from the leaves of Calotropis gigantea, Adhatoda vasica and Artocarpus integrifolia on human whole blood and peripheral blood mononuclear cells (PBMC) against hepatitis B vaccine containing surface antigen (HBsAg). Methods: for these studies, our group evaluated variable doses of fl avonoids extracted from the leaves of Calotropis gigantea, Adhatoda vasica and Artocarpus integrifolia and estimated blood counts (lymphocytes, monocytes and granulocytes count using fl ow cytometry); nitric oxide production (using Griess reagent assay) and proliferation assay against HBsAg. Results: The results of these studies claimed that these fl avonoids showed sudden decline in monocytes and granulocytes count; nitric oxide production and also showed its cytotoxicity in the form of proliferation at higher doses as compared to control. Conclusion: Overall, these fl avonoids showed immunosuppressive as well as cytotoxic effect against HBsAg and also probable candidate for anticancer agent. Research Article Immunosuppressive and Cytotoxic Potential of Flavonoids from Medicinal Plants: Preliminary Investigation for Anticancer Activity Amit Gupta* and Revati N Chavan Department of Immunology and Virology, Vidya Pratishthan’s School of Biotechnology (VSBT) Research Centre, Affi liated to Savitribai Phule Pune University, Maharashtra, India Dates: Received: 05 December, 2016; Accepted: 30 January, 2017; Published: 01 February, 2017 *Corresponding author: Amit Gupta, Doctor, Assistant Professor, Department of Immunology and Virology, Vidya Pratishthan’s School of Biotechnology (VSBT), Research Centre, Affi liated to Savitribai Phule Pune University, Baramati, Maharashtra, India, E-mail:


Introduction
Cancer is one of the major public health concern in all over the world. Several medicinal plants were screened and determined some anti-cancer agents i.e. taxol, vinblastine, vincristine, campothecin derivatives etc. Other promising anticancer agents that are reported i.e. fl avopiridol, roscovitine, betulinic acid, silvestrol etc., [1]. In addition, there is another group that derived from medicinal plant products in the form of secondary metabolites i.e. fl avonoids and their synthetic analogs are generally used in the treatment of ovarian, breast, cervical, pancreatic and prostate cancer [1][2][3].
Flavonoids (formed from the aromatic amino acids i.e. phenylalanine, tyrosine, and malonate) are one of the important constituent in the human diet, although they are generally considered as non-nutrients [4]. Generally, these fl avonoid intakes can range between 50 and 800 mg a day, depending on the diet -consumption of vegetables, fruit, red wine, tea, unfi ltered beer, etc. The absorption of the dietary fl avonoids liberated from the food by chewing will depend on its physicochemical properties (i.e. molecular size, confi guration, lipophilicity, solubility etc.) [4][5][6].
Generally, these fl avonoid should be absorbed in small intestine or has to go to the colon before absorption and also depends upon the structure of fl avonoid. Most fl avonoids, except for the subclass of catechins, are present in medicinal plants but they are actually bound to sugars as b-glycosides.
As per the literature, fl avonoid glycosides converted into aglycan form and they are easily absorbed in small intestine Citation: Gupta  are transported across the small intestine by the intestinal Na+-dependent glucose cotransporter (SGLT1). More than 4000 varieties of fl avonoids isolated from various medicinal plants which is already identifi ed. The most important effect of these fl avonoids is the scavenging of oxygen-derived free radicals that is reported. In vitro experimental systems also showed that fl avonoids possess anti-allergic, anti-viral and anti-carcinogenic properties [7,8]. The fl avonoid is bound to albumin and then transported to the liver. The liver can extend the conjugation of the fl avonoid by adding sulfate group, methyl group, or both. The addition of these groups which increases the circulatory elimination time and probably also decreases its toxicity. According to the literature, lot of fl avonoids are reported from these medicinal plant products e.g. only one fl avonoid was isolated from the leaves of Juniperus procera and could be identifi ed as; 3′,4′,3,7-tetrahydroxyfl avone [9], new fl avonoid were also isolated from the stem bark of Choerospondias axillaries, the fruit of which was used for the treatment of cardiovascular diseases [10] etc.
Adhatoda vasica (Adulsa; family Acanthaceae), medicinal plant products especially leaves showed various medicinal properties and it will protect our immune system against number of infectious diseases [13,14] e.g. cold, cough, asthma, malaria,  [15]. This is the fi rst preliminary study related to anticancer activity of fl avonoids isolated from medicinal plants especially leaves in order to determined its immunomodulatory (immunostimulator/immunosuppressor) effect against specifi c protein antigen.

Estimation of blood counts
For these studies, exposure of variable doses of fl avonoids
In cytotoxicity assay, similar volume of fresh medium were added and again incubated the plate for another 24 h. After incubation, add MTT (5 mg/ml; 10 μl) solution and again incubate it for another 3-4 h. Thereafter, centrifuging the plate, discard the supernatant and appearance of formazon crystals settled at the bottom. These crystals were dissolved in dimethyl sulphoxide and the optical density was measured at 570 nm [11][12][13][14].
For these studies, PBMC cell culture supernatant was mixed with similar volume of Griess reagent (1% sulfanilamide and 0.1% naphthylethylene diamine dihydrochloride in 2.5% phosphoric acid) and incubated the plates at room temperature for another 10 minutes and absorbance at 540 nm was measured by spectrophotometer. The fresh culture medium (RPMI containing 10 % fetal bovine serum) was used as a blank. The nitrite quantity was determined from a sodium nitrite standard curve.

Statistical analysis
Values are expressed as Mean ± S.E. The difference between control and fl avonoids extracted from Calotropis gigantea,

Adhatoda vasica and A. integrifolia which is determined through
Bonferroni multiple comparison test (one way ANOVA test).

Estimation of lymphocytes, monocytes and granulocytes count
The result of these studies showed that these fl avonoids extracted from the leaves of Calotropis gigantea, Adhatoda vasica and Artocarpus integrifolia showed dramatically decreased in monocytes as well as granulocytes count as compared to control (Figure 1). In other words, these fl avonoids showed immunosuppressive and cytotoxic effect at higher doses against specifi c protein antigen.

Cytotoxicity assay
The effect of variable doses of fl avonoids on determining its cytotoxicity in human PBMC's against specifi c protein antigen i.e. HBsAg as shown in Figure 2. The results of these studies showed that these fl avonoids showed declined in proliferation at higher doses after treating with PBMC as compared to control.

Nitric oxide production
The effect of variable doses of fl avonoids on nitric oxide production in human PBMC's as shown in Figure 3. The results showed that these fl avonoids showed declined in nitric oxide at higher doses after treating with PBMC as compared to control. As per the literature survey, consumption of isofl avones are directly associated with decreased risk of estrogen related cancers and vascular diseases whereas total fl avonoids including fl avanones, and fl avonols were inversely proportional to oral and laryngeal cancers [18,19]. In addition, reduced risk rate of colorectal cancer was detected in high intake of fl avones, isofl avones, anthocyanidins and fl avonols. In contrast, there is no association between fl avonoids and prostate cancer reported or emerged whereas inverse association was found between proanthocyanidins and colorectal cancer [20]. In this regard, we focused on various medicinal plants pertaining to fl avonoids isolated from medicinal plants and determined its immunopharmacological activity against specifi c protein antigen.

Discussion
Modulation (stimulatory or suppressive) of the immune Adhatoda vasica C) Artocarpus integrifolia in order to lymphocytes, monocytes and granulocytes count in human whole blood which is exposed with HBsAg (20 μg/ml; 10 μl). After treatment with fl avonoids, lysed the cells with red cell lysis buffer and wash the cells with phosphate buffered saline and analyzed the samples (10000 events, cell quest software) through fl ow cytometer.
Citation: Gupta  As per literature, nitric oxide production is mainly released by human PBMC and played a considerable role especially in the pathophysiology of hormonal immune system [21]. Actually, diseases related to animal or human can be characterized or diagnosed through lack or excess of nitric oxide production from human PBMC or animal peritoneal macrophages. In some circumstances, protection against decline in constitutive nitric oxide production or stimulation in the vasculature may intercept the development of vascular disease, while inhibition of uncontrolled nitric oxide production could also be a therapeutic target [21]. From these studies, these medicinal plants i.e.

Calotropis gigantea, Adhatoda vasica and Artocarpus integrifolia
showed inhibition of proliferation and nitric oxide production at higher doses as compared to control. In other words, these fl avonoids showed some drastic decreased in nitric oxide production at higher doses and showed its therapeutic effect.
In addition, these fl avonoids of these medicinal plants showed inhibitory effects on HBsAg lymphocyte proliferation. From these results, it is confi rmed that fl avonoids from Calotropis gigantea, Adhatoda vasica and Artocarpus integrifolia showed immunosuppressive and cytotoxic effect against HBsAg.

Conclusion
The present study has shown the immunosuppressive and cytotoxic potential of fl avonoid extracted from the leaves of Calotropis gigantea, Adhatoda vasica and Artocarpus integrifolia by inhibiting cellular immunity in the form of nitric oxide production, blood counts and proliferation assay. These fi ndings also suggested that these fl avonoids from medicinal plants suppress the immune system and also be one of the probable candidate like other chemotherapeutic agents. Further detailed studies of these fl avonoids are required which might establish a possible mechanism for anticancer activity.