INTRODUCTION:

Plants are used medicinally in several countries which plays a major role in different sources such as in pharmacology, cosmetics, dyeing industries, nutraceuticals and also a source of many potent and powerful drugs¹. Many medicinal plants and wild edible plants have been studied for their invaluable source of bioactive compounds which could be utilized for therapeutic purposes to reduce side effects. Alkaloids, tannins, flavonoids and phenol are important bioactive compounds and are widely utilized in human therapy, agriculture, vetinary, research project and countless other areas².

R. communis is one among the traditional medicinal plant contains active secondary metabolites which have direct or indirect use in the treatment of various human diseases^3,4.

Ricinus communis belongs to the family Euphorbiaceae. It is a fast-growing perennial shrub usually grows in waste lands. The plant is commonly known as ‘castor plant’ because the seeds of this plant is widely used for castor oil production. The extracted oil helps to cure indigestion and possess antimicrobial properties^5,6. It is also used in the manufacture of lubricant, cosmetics, soaps, printing ink, plastics, varnishes, paints, textile dyes, waxes, fungicides and in the production of ointment bases^7,8. The leaf extracts possess antioxidant, antifungal, insecticidal, larvicidal and wound healing activities⁹. Thus, the present study deals with the screening based on phytochemical analysis for identifying the chemical constituents of R. communis.

MATERIALS AND METHODS:

Sample Collection:

Fresh and healthy leaflets of Ricinus communis were collected from Dakshina Kannada district, Karnataka during the month of December 2015. The plant materials were identified and authenticated by the taxonomist, Department of Botany, Mangalore University. The plant specimens have been preserved in the laboratory for further use. The collected materials were washed with running tap water to get rid of dust particles and rinsed with distilled water. The leaves were shade dried and ground into fine powder. The ultimate uniform powder was used for methanolic extract preparation.

Extract Preparation:

20 g of finely pulverized sample was macerated with 200 mL of methanol and the flask was kept on an orbital shaker (190-220 rpm) for 48 hours at room temperature. The filtrate was separated after 48 hours using Whatman No. 1 filter paper. The residue was macerated again using methanol for complete extraction. The crude extract was then kept at 4°C until the experiment was completed.

Qualitative Phytochemical Analysis:

Qualitative analysis of metabolites such as tannins, saponins, alkaloids, flavonoids, terpenoids, glycosides, steroids, phenols, carbohydrates, protein, phlobatannins, and coumarins were carried out on crude methanolic extract according to the standard procedures^10-13.

Quantitative Analysis:

Total Flavonoid Content:

0.5 mL of the sample was mixed with 1.5 mL methanol, 0.1 mL of 10 % AlCl₃, 0.1 mL of 1M potassium acetate, and 2.8 mL of distilled water. After 30 min, of incubation at room temperature, the absorbance of the reaction mixture was measured at 415 nm. The amount of flavonoid content was expressed as (μg) equivalents of quercetin/mg of sample^14,15.

Total Tannin Content:

The tannin content was estimated by method¹⁶ with slight modifications. 20 μL of the sample was aliquoted into a test tube containing 980 μL of distilled water. To this, 500 μL of 1 % potassium ferricyanide (K₃Fe (CN)₆) and 100 μL of 1 % ferric chloride (FeCl₃) were added and made up to 3 mL with distilled water. After 10 min, the reaction mixture was measured by employing a UV spectrophotometer at 720 nm. The tannin content was expressed as μg of tannic acid equivalents/mg of extract.

Determination of Alkaloid:

200 mL of 10 % acetic acid in ethanol was added to 2.5 g of powdered plant sample in a 250 mL beaker and covered with aluminum foil. The reaction mixture was allowed to stand for 4 hours. The extract was concentrated on a water bath to one-quarter of the original volume followed by the addition of concentrated ammonium hydroxide dropwise to the extract until the precipitation was complete. The supernatant was discarded and the precipitates were collected, washed with dilute ammonium hydroxide, and filtered. The obtained residue was dried in an oven and weighed¹⁷.

Percentage of alkaloid was determined using the subsequent formula:

Percentage of alkaloid = (final weight of the sample/initial weight of the extract) × 100

Total Phenols:

The total phenolic content present in the extract was determined using Folin–Ciocalteu (FC) method^18,19. 0.1 mL of sample was made up to 0.25 mL with distilled water and mixed with 0.25 mL of FC phenol reagent. After 3 min, 0.5 mL of 20 % sodium carbonate solution was added to the mixture and made up to 5 mL by adding distilled water. The resultant mixture was kept in the dark for 30 min, then the absorbance was read at 760 nm using the spectrophotometer. The results were expressed as μg of gallic acid equivalents/mg of extract.

Antioxidant Activity:

Total Antioxidant Activity:

The reduction capacity of methanolic plant extract of R. communis was assessed by phosphomolybdenum method according to the procedure described²⁰. The reagent solution was prepared using 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. Test tubes containing 0.3 mL of the extract with 3 mL of reagent solution were incubated at 95°C for 90 min. Then, the absorbance of the colored complex was measured at 695 nm using a UV-VIS spectrophotometer against blank after cooling to room temperature. Methanol (0.3 mL) in the place of the extract was used as the blank. The total antioxidant activity was expressed as the number of gram equivalent of ascorbic acid.

DPPH (1, 1diphenyl-2-picryl hydrazyl) Radical Scavenging Assay:

DPPH free radical scavenging assay was measured by method²¹. Various concentrations (20 -100 μg/mL) of a leaf extract (2 mL) were taken in several vials containing 3 mL of 0.1 mM methanolic solution of DPPH. The test tubes were shaken gently and kept aside for 30 min at room temperature in the dark. An optical density of the sample was measured at 517 nm against blank. Ascorbic acid was used as the standard control. All the tests were performed in triplicates.

Free radical scavenging activity was expressed as inhibition percentage and was calculated using the following formula:

Percentage of inhibition (%) = (OD of control – OD of sample)/ OD of control X 100

Reducing Power Assay:

Different concentrations of the extract were prepared in methanol solvent and assorted with 2.5 mL of 0.2 M phosphate buffer followed by 2.5 mL of freshly prepared 1 % potassium ferricyanide (K₃Fe (CN)₆). This mixture was incubated for 20 min at 50°C. To this, 2.5 mL of 10 % trichloroacetic acid was added and centrifuged at 3000 rpm for 10 min. 2.5 mL of the clear extract was assorted with 2.5 mL of methanol and 0.5 mL of 0.1 % FeCl₃. The absorbance was measured at 700 nm. The experiment was conducted in triplicates, and the reducing power was expressed as (mg/g) equivalents of ascorbic acid of the extract²².

ABTS Radical Scavenging Assay:

Free radical scavenging activity of plant samples was determined by ABTS radical cation decolorization assay²³. ABTS⁺ cation radical was produced by the reaction between 7 mM ABTS in water and 2.45 mM potassium persulfate (1:1), stored in dark at room temperature for 12-16 hrs. before use. After the addition of 10 μL of plant extract to 1 ml of ABTSradical solution, the absorbance was measured at 734 nm. An appropriate solvent blank was run in each assay. All the measurements were carried out at least three times. Percentage of inhibition was calculated using the formula,

ABTS⁺ scavenging activity (%) = (Ab–Aa) / Ab) ×100

Where Ab is the absorbance of ABTS radical + methanol; Aa is the absorbance of ABTS radical + sample extract.

GC–MS Analysis:

Methanolic extract of R. communis was subjected to GC-MS analysis [GC-MS model-GCMS-TQ8040NX, Japan, 30 m long, 0.25 mm thick SH-Rxi-5Sil MS column and internal diameter of 0.25 mm (5 % diphenyl /95 % dimethylpolysiloxane)]. The column oven temperature was set to 60.0°C and the injector temperature was 260.00°C and helium (99.999 %) was acted as carrier gas at a flow rate of 1.20 mL/min. 1.0 µL of each extract was injected by the split less injection technique and split ratio of 10.0 was used for the analysis. Mass ranges were scanned at a rate of 3333 scans/0.200 seconds from 40 to 650 m/z and the total GC run time was maintained to 40 min. The mass spectra of the compounds found in the extracts were matched with the National Institute of Standards and Technology (NIST) 17 libraries²⁴.

RESULTS:

Phytochemical Analysis:

Qualitative analysis of leaf extracts showed the presence of phytoconstituents viz., alkaloids, tannins. flavonoids, saponins, steroids, coumarins, but terpenoids, phlobatannins, and glycosides were absent (Table 1).

Table 1: Qualitative analysis of phytochemicals of R. communis L.

Phytochemicals	Reagents / Chemicals	Observation	Results
Alkaloids	Dragendorff’s reagent	Orange-red color	+
Flavonoids	Lead acetate	Yellow color	+
Tannins	Ferric chloride	Green color	+
Saponins	Distilled water	Frothing seen	+
Terpenoids	Chloroform and H₂SO₄	Greyish color not observed	-
Steroids	Liebermann‑Burchard test	Reddish ring	+
Phlobatannins	HCl	Red precipitate not observed	-
Glycosides	HCl, Sodium nitroprusside, and Sodium hydroxide	Pink-red color not observed	-
Coumarins	NaOH	Yellow coloration	+
Phenols	Ferric chloride	Dark green color	+

(+) = Present, (-) = Absent

Quantitative Analysis:

Total flavonoid, tannin, alkaloid, and phenol:

A calibration curve was made with the absorbance (at 415 nm) of the quercetin solutions in methanol to calculate the flavonoid content of the extract. The mean value of the content of flavonoids in the extract was 256.29 ± 0.21 mg QE/g. Tannin content was estimated using tannic acid standard and expressed as tannic acid equivalents. R. communis leaf extract showed a tannin content of 175.59 ± 0.78 mg TAE/g and the total alkaloid content of the extract was found to be 32.28 ± 0.28 mg/g. The total phenolic content was measured by Folin- Ciocalteu method and was expressed as mg gallic acid equivalents (GAE) per gram of the plant material. The phenolic content obtained from the methanolic extract of R. communis was 273.35 ± 2.33 mg GAE/g (Table. 2).

Table 2. Quantitative analysis of methanolic leaf extracts of R. communis L.

Phytochemicals	Results
Flavonoid (mg QE/g)	256.29 ± 0.21
Tannin (mg TAE/g)	175.59 ± 0.78
Alkaloid (mg/g)	32.28 ± 0.28
Phenolics (mg GAE/g)	273.35 ± 2.33

Antioxidant Activity:

Four different methods such as total antioxidant (phosphomolybdenum), DPPH radical scavenging activity, reducing power assay and ABTS methods were used to assess the antioxidant activity of R. communis and the results were presented in Table 3. In DPPH and ABTS assay results were expressed in percentage of inhibition (%) and the extract showed strong scavenging activity to DPPH. Total antioxidant activity of R. communis extract by phosphomolybdenum method was found to be higher compared to reducing power assay and the results were expressed in mg/g.

Table 3. Antioxidant activity of methanolic leaf extracts of R. communis L.

Method	Activity
DPPH (%)	84.22 ± 0.81
ABTS (%)	58.75 ± 0.52
Reducing Power (mg/g)	92.12 ± 1.09
Total antioxidant (mg/g)	385.72 ± 0.84

GC MS Analysis:

The GC–MS chromatogram of methanolic extract of R. communis leaves showed 23 peaks (Fig. 1) with different retention time. Peak area percentages less than 1 % are not presented here. The results obtained were identified after comparing the mass spectra with those of NIST17 libraries. The identified compounds and their retention time, peak area percentage, molecular formula, and molecular weight are presented in Table 4.

Table 4. Phytoconstituents identified in the methanolic leaf extract of R. communis L. by GC–MS analysis.

Sl No.	Retention time	Compound name	Peak area %	Molecular formula	Molecular weight
1	3.696	N-Methoxy-N-methylacetamide	5.99	C₄H₉NO₂	103.12
2	3.850	Glycerine	1.19	C₃H₈O₃	92.09
3	8.342	4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy	1.16	C₆H₈O₄	144.12
4	10.726	3-Allyl-6-methoxyphenol	6.64	C₁₀H₁₂O₂	164.20
7	17.063	Neophytadiene	2.74	C₂₀H₃₈	278.51
8	17.269	2,3a-Dimethylhexahydrobenzofuran-7a	1.33	C₁₀H₁₈O₂	170.24
9	17.949	Hexadecanoic acid, methyl ester	1.63	C₁₇H₃₄O₂	270.45
10	18.306	n-Hexadecanoic acid	5.78	C₁₆H₃₂O₂	256.4
11	18.634	Ricinine	11.96	C₈H₈N₂O₂	164.16
12	19.635	9,12,15-Octadecatrienoic acid, methyl ester	2.21	C₁₉H₃₂O₂	292.5
13	19.739	Phytol	9.20	C₂₀H₄₀O	296.5
14	19.984	cis, cis, cis-7,10,13-Hexadecatrienal	2.12	C₁₆H₂₆O	234.38
15	23.713	Hexadecanoicacid,2-hydroxy-1-hydroxymethyl	1.28	C₁₉H₃₈O₄	330.5
16	29.715	28-Norolean-17-en-3-ol	1.94	C₂₉H₄₈O	412.69
17	30.816	Vitamin E	1.66	C₂₉H₅₀O₂	430.71
18	33.306	Stigmasterol	6.77	C₂₉H₄₈O	412.69
19	34.754	Gamma. -Sitosterol	5.64	C₂₉H₅₀O	414.7
20	35.767	28-Norolean-17-en-3-one	3.92	C₂₉H₄₆O	410.67
21	36.585	28-Norolean-17-en-3-ol	3.85	C₂₉H₄₈O	412.69
22	36.858	9,19-Cyclolanost-24-en-3-ol, (3. beta.)-	1.48	C₃₂H₅₂O₂	468.76
23	37.036	Lupeol	1.57	C₃₀H₅₀O	426.7

Fig.1: GC - MS chromatogram of methanolic extract of R. communis

DISCUSSION:

The plants and their chemical compounds constitute the principal source of microbicide, pesticides, and many pharmaceutical drugs²⁵. The qualitative analysis of R. communis revealed the presence of major compounds which are known to exhibit medicinal properties²⁶. The quantification of the phytochemicals is necessary for drug formulation²⁷. This research has been shown that an appreciable quantity of phenols, flavonoid, alkaloid, and tannin content is present in the methanolic extract of R. communis. Flavonoids act as a potent water-soluble antioxidant and inhibit the progression of tumor cells, reduction of coronary heart diseases, and significant inhibitory potential against a wide range of enzymes^28,29,30. Tannins are a group of phenolic compounds and have a major role in plant defense mechanisms against fungal and bacterial pathogens³¹. They contribute the quality and nutritional value in terms of modifying color, taste, aroma and also in providing health beneficial effects³². Plant-derived alkaloids are low molecular weight, nitrogen containing compounds in clinical use for the production of anticancer agents, muscle relaxants, sedative agents, and stimulants³³. Thus, total phenolic content, flavonoid, alkaloid, and tannin contents were evaluated. Antioxidants are important substances that can protect the body from damage due to free radical induced oxidative stress³⁴. Consumption of dietary supplements containing plant antioxidants helps to prevent the creation of various diseases and disordors³⁵. The antioxidant potential of R. communis methanol extract was investigated and was found that the plant contains a rich source for bioactive compounds which are capable to reduce oxidative stress. Most of the polyphenols from plants also act as reducing agents and antioxidants by the hydrogen donating property of their hydroxyl groups³⁶. So, the presence of polyphenols might be responsible for the antioxidant activity.

A combination of gas chromatography and mass spectrometry is a direct and sophisticated approach for the separation, identification, and extraction of individual components from their mass spectra³⁷.

The compounds identified in the crude methanolic extract of R. communis are N-Methoxy-N-methylacetamide, Glycerine, 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy, 3-Allyl-6-methoxyphenol, Neophytadiene, 2,3a-Dimethylhexahydrobenzofuran-7a, Hexadecanoic acid, methyl ester, n-Hexadecanoic acid, Ricinine, 9,12,15-Octadecatrienoic acid, methyl ester, Phytol, cis, cis,cis-7,10,13-Hexadecatrienal, Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl, 28-Norolean-17-en-3-ol, Vitamin E, Stigmasterol, .gamma.-Sitosterol, 28-Norolean-17-en-3-one, 28-Norolean-17-en-3-ol, 9,19-Cyclolanost-24-en-3-ol, (3.beta.)- and Lupeol. Among them, the compounds with a higher percentage of peak are Ricinine (11.96 %), Phytol (9.20 %), 3-Allyl-6-methoxyphenol (6.64 %), Stigmasterol (6.77 %), N-Methoxy-N-methylacetamide (5.99 %), gamma. - Sitosterol (5.64 %), n-Hexadecanoic acid (5.78 %). Ricinine is a poisonous alkaloid present in the leaves and seeds of the castor plant. Various studies showed that ricinine is a toxic compound effect on the central nerve system³⁸. As per the investigation of Carolina 2019³⁹, GCMS analysis of methanolic extract of R. communis leaves contains ricinine was the main content and the addition of castor extracts to the Aspergillus niger resulted in the inhibition of its cell biomass growth. Ricinine was found to be active against some nematodes⁴⁰. Phytol was observed to be the second main component in GCMS analysis. It is a precursor for vitamins E and K1 and a preventive agent against breast cancer cells⁴¹. Studies revealed the antimicrobial activity of phytol against E. coli, C. albicans, and A. niger with no remarkable toxicity and were stable on stone, MDF and steel until 36 hours. So, phytol is a good choice for the disinfection of surfaces⁴². Other identified compounds from R. communis are also possessed biological activity and responsible for the use of this plant for various pharmacological purposes.

CONCLUSION:

Medicines derived from plants have made immense contributions towards the betterment of human health and act as a source of inspiration for novel drug compounds. From the above research, it can be concluded that this plant has enormous potential to be used in the area of pharmacology and as a prospective source of valuable drugs. Due to the presence of various compounds that are essential for good health, they can also be used to improve the health status of society. A spectrum of compounds identified by various analyses in this study act as a strong antibacterial, antifungal, antioxidant, and anti-inflammatory activities.

ACKNOWLEDGEMENT:

The authors are thankful to the Department of Biosciences and DST PURSE laboratory, Mangalore University, Mangalagangothri, Karnataka, India, for providing laboratory facilities to carry out the work.

CONFLICT OF INTEREST:

We declare that we have no conflict of interest.

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Received on 28.08.2021 Modified on 26.02.2022

Research J. Pharm. and Tech 2022; 15(12):5689-5694.

DOI: 10.52711/0974-360X.2022.00959