*3.3. Fractionation of Aqueous Ethanolic Extract of P. maderaspatensis*

Fractionation of the most potent aqueous ethanolic extract into hexane soluble, ethyl acetate sediment, ethyl acetate soluble, chloroform sediment, chloroform soluble, methanol, and water-soluble fractions was carried out. These fractions were studied for their TLC pattern using the above-mentioned solvent systems. The data regarding the number of spots and their resolution are given in Table 1.

The solvent system no.7 indicated the best separation of polyphenols in the ethyl acetate fraction. Except for the ethyl acetate fraction, none of the other fractions indicated the presence of selected polyphenols. Other fractions were discarded. The ethyl acetate fraction was selected for vacuum liquid chromatography.

**Figure 1.** Simultaneous HPTLC profile of hydroethanolic extracts of *P. maderaspatensis* with different markers in the solvent system: toluene/ethyl acetate/formic acid/methanol (3:3:0.08:0.02) (*v*/*v*) at 254 nm.

**Table 1.** TLC analysis of ethyl acetate fraction using different solvent systems and visualising agents.


#### *3.4. Stimulation of Inducible NO Synthesis by the Different Fractions of P. maderaspatensis*

Ethyl acetate, chloroform, hexane, and water-soluble fractions, along with sediment of chloroform and ethyl acetate, were then screened at different concentrations (25, 50, and 100 μg mL<sup>−</sup>1) for invitro immunomodulatory activity using the nitric oxide assay method. Hexane, chloroform, and sediment between chloroform and water-soluble fractions had no significant effect on LPS-stimulated NO production by the RAW 264.7 cells, while chloroform sediment (25 μg mL−1) and ethyl acetate fraction at concentrations of 25 and 50 μg mL−<sup>1</sup> had a significant inhibitory effect on LPS-stimulated NO production when compared with LPS control. The ethyl acetate fraction and chloroform fraction (100 μg mL<sup>−</sup>1) had a more significant stimulatory effect on LPS-stimulated NO production by the RAW 264.7 cells, as represented in Table 2 and Figure 2.

**Table 2.** Effect of *P. maderaspatensis* fractions on NO production in LPS-stimulated RAW 264.7cells.


**Figure 2.** Effect of various fractions based on polarity on LPS-stimulated RAW 264.7 cells. Cells in 96-well plates (1 × 106 cells/well) were first incubated with and without specified concentrations of crude extracts for 2 h, and then incubated with LPS (10 μg mL<sup>−</sup>1) for 20 h. ## LPS treated. Untreated is the negative control without LPS treatment. Each value was expressed as mean ± SEM in the triplicate experiment. ns: non-significant, *n* = 3, data ± S.E.M. Groups 3–7 were compared against group II using Dunnett's post-hoc test (\* significant at < 0.01; \*\* significant at < 0.001).

*3.5. Vacuum Liquid Chromatography and Selected Activity of Column Eluents of Ethyl Acetate Fraction*

Fourteen column eluents were obtained from the column chromatography of the ethyl acetate fraction of *P. maderaspatensis*. These column eluents were subjected to the HPTLC profile. Different components present in the eluents were identified by spraying with NP reagent and subsequently matching the Rf value with the standards (1–6). The detailed HPTLC profile of each fraction is represented in Table 3.

The standards (1–6) were identified as follows: ellagic acid (Rf value: 0.55) standard was matched with track no. 19 (100% ethyl acetate elutes), eupalitin(Rf value: 0.23) standard was not matched with any track, kaempferol (Rf value: 0.81) standard was matched with track no. 8 (25% ethyl acetate in toluene elutes), rutin (Rf value: 0.08) standard was matched with track no. 13 (50% ethyl acetate in toluene 3rd elutes), epicatechin (Rf value: 0.50) standard was matched with track no. 28 (75% ethyl acetate in toluene elutes), epicatechin (Rf value: 0.50) standard was matched with track no. 15 (75% ethyl acetate in toluene elutes), and catechin (Rf value: 0.54) standard was matched with track no. 15 (75% ethyl acetate in toluene elutes).

### *3.6. Effect of Column Eluents of Ethyl Acetate Fraction on LPS-Stimulated NO Production in RAW 264.7 Cells*

The effect of nine column eluents of ethyl acetate fractions on NO production was determined by treating the RAW cell of LPS stimulation/inhibition by pre-incubating the cells with or without the elutes. LPS significantly increased NO production in RAW 264.7 cells. The levels of NO production induced by LPS-stimulated were significant (\*\* *p* < 0.01) in a dose-dependent manner when treated with concentrations of 25 and 50 μg mL−<sup>1</sup> of each elute and significantly stimulated by column eluents of ethyl acetate fractions.NO production was 25 and 50 μg mL−<sup>1</sup> of column eluents of ethyl acetate fractions of *P. maderaspatensis* compared with LPS treatment alone. In this study, a comparison of

column elutes of ethyl acetate fractions was carried out in mouse monocyte cell lines RAW 264.7 cells, as shown in Figure 3.

**Table 3.** HPTLC analyses of column eluent of ethyl acetate fractions of *P. maderaspatensis* using VLC.


**Figure 3.** Effect of column eluents of ethyl acetate fractions on NO levels in LPS-stimulated RAW 264.7 cells. Cells (1 × 106 cells/well) in 96-well plates were first incubated with and without specified concentrations of column eluents for 2 h, and then incubated with LPS (10 μg/mL) for 20 h. ## LPS treated. Untreated is the negative control without LPS treatments. Values are expressed as mean ±SEM. ns: non-significant, *n* = 3, data ± S.E.M. Groups 3–7 were compared against group II using Dunnett's post-hoc test (\* significant at < 0.01; \*\* significant at < 0.001).

#### *3.7. Stimulation of Inducible NO Synthesis by Compounds*

The significant suppressive effect by concentration at 50 μg mL−<sup>1</sup> and 100 μg mL−<sup>1</sup> of rutin, kaempferol, gallic acid, and ursolic acid; the minimum concentration of ellagic acid (50 μg mL<sup>−</sup>1); and the more significant stimulatory effect of oleanolic acid, ellagic acid, and quercetin in LPS stimulated NO production by the RAW 264.7 cells were observed (Figure 4).

The compounds indicated significant invitro immunomodulation: ellagic acid > quercetin > oleanolic acid and immunosuppressive effect: kaempferol > catechin > rutin > gallic acid > ellagic acid (50 μg mL<sup>−</sup>1), when compared with LPS.

**Figure 4.** Effect of polyphenols on NO levels in LPS-stimulated RAW 264.7 cells. Cells (2 <sup>×</sup> <sup>10</sup><sup>4</sup> cells /well) in 96-well plates were first incubated with and without indicated concentrations of polyphenols for 2 h and then incubated with LPS (10 μg mL<sup>−</sup>1) for 20 h. ## LPS treated. Untreated is the negative control without LPS treatments. Values are expressed as mean ± SEM. ns: non-significant, *n* = 3, data ± S.E.M. Groups 3–7 were compared against group II using Dunnett's post-hoc test (\* significant at < 0.01; \*\* significant at < 0.001).

#### **4. Discussion**

A single solvent system (toluene/ethyl acetate/formic acid/methanol (3:3:0.8:0.2 *v*/*v*) was developed for densitometric quantification of polyphenols by HPTLC in aqueousalcoholic extracts with reference to respective marker compounds such asrutin, kaempferol, quercetin, catechin, ellagic acid, gallic acid, and quercetin present in *P. maderaspatensis.* Ethyl acetate supernatant fractions showed significant immunomodulatory activity compared with other fractions, and we subsequently separated ethyl acetate fraction by vacuum liquid chromatography. Using ethyl acetate fraction in *P. maderaspatensis*, we performed isolation, purification, and characterization of rutin, kaempferol, ellagic acid, gallic acid, and catechin. Ellagic acid showed more significant immunomodulatory activity, while quercetin exhibited significant immunomodulation when compared with the crude. Specifically, ethyl acetate (100%) column eluents indicated that ellagic acid exhibited significant immunomodulation, followed by 15% methanol elutes, followed by chloroform second sediment fraction (oleanolic acid and ursolic acid), followed by 75% ethyl acetate(catechin, epicatechin) and 50% ethyl acetate elutes showed highly significant suppression (rutin, gallic acid), followed by 25% ethyl acetate (kaempferol and quercetin) and 75% methanol indicated non-significant active fraction, followed by 75% ethyl acetate second elutes and chloroform first fraction when compared with LPS.

Synthetic agents, natural adjuvant, and antibody reagents are used as immunosuppressive and immunostimulants. However, there is a major limitation to the general use of these agents, that is, the increased risk of infection and generalized effect throughout the immune system [26]. Many therapeutic effects of plant extracts have been claimed to be thanks to their influence on the immune system of the human body [27]. Many herbal

preparations such as *Panax ginseng*, *Picrorhiza scrophulariiflora*, *Centella asiatica*, *Tinospora cordifolia*, *Phyllanthus debilis*, *Trigonella foenum graecum*, and *Pouteria cambodiana* have been shown to alter the immune function and report a wide array of immunomodulatory effects [28–34]. Most research concerning the immunomodulatory activities of the plant has been carried out using crude extracts [35,36]. In some, combinations of various herbs or herbs in combination with minerals have been used, taking into consideration Unani, Ayurvedic, or Chinese traditional formulation. Although it may be rational to use a single plant or its single constituents, it has been a general experience that the single constituent shows more efficacy compared with the total plant extract. *Phyllanthus* genus was found to be rich in polyphenols, lignins, flavonoids, triterpenes, hydrolysable tannins, sterol, and alkaloids [37].

## **5. Conclusions**

We evaluated the immunomodulatory activity of various fractions, column eluents of ethyl acetate fraction, and their polyphenols present in *P. maderaspatensis* obtained from the Maruthmallai region of Kanyakumari district, Tamilnadu, India. Rutin, gallic acid, kaempferol, and catechin, each 200 μg mL<sup>−</sup>1, as well as ellagic acid (100 μg mL<sup>−</sup>1), showed a significant immunosuppressive effect owing to the inhibition of NO production compared with the LPS-stimulated RAW 264.7 cells group, and the most significant immunostimulatory effect was produced by ellagic acid and quercetin, each 200 μg mL<sup>−</sup>1, when compared with the LPS control group.

**Author Contributions:** Conceptualization, U.I., D.P.K. and V.A.; methodology, U.I., D.P.K. and V.A.; investigation, U.I.; data curation, U.I., D.P.K., V.A., P.P.N. and M.E.-M.; writing—original draft preparation, U.I., D.P.K., V.A. and P.P.N.; writing—review and editing, U.I., D.P.K., V.A., P.P.N., M.E.- M. and M.S.K.; visualization, U.I.; supervision, V.A.; funding acquisition, U.I., D.P.K., V.A., P.P.N., M.E.-M. and M.S.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors extend their appreciation to the Deanship of scientific research at king Khalid University, Grant No: RGP2/191/42 Saudi Arabia for financial assistance.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank the National Medicinal Plant Board, Department of AYUH, Delhi, India.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

### **References**

