INTRODUCTION:

In the study of pharmacological substances, natural substances originating from medicinal plants are useful resources¹. The creation of novel medications from medicinal plants has been greatly aided by the technical advancements in science that have made it easier to isolate, identify, and elucidate chemical principles from natural sources².

Over 7,000 plants have been identified as being used for medicinal and aromatic reasons in India. The usage of medicinal herbs in India has a long history stretching back to pre-Vedic times, at least 4,000 years³. Currently, it is estimated that at least 70% of the nation's population gets their basic medical care from herbal remedies, and many more use them in addition to other types of therapy⁴.

Many significant medications and physiologically active compounds have been produced by traditional medicinal plants. Corchorus (Family Tiliaceae) has almost 100 species that are found in tropical and subtropical environments, mostly in South Asia as well as South America⁵. Corchorus depressus (Linn.), or "Bhedani" in Sanskrit, "Bauphali" in Hindi, and "Malukh" in Arabic, is an herb that grows in dry regions of India, especially Odisha, Punjab, Gujarat, and Rajasthan⁶. It's a perennial plant with prostrate growth and deep roots and branches. Flowers are yellow in color, and leaves are plicate⁷.

Fig.1 Leaves and flowers of Corchorus depressus (Linn.)

Its extract is used for the treatment of gonorrhea and utilized as a paste for soothing injuries. The perennial plant serves as a cooling remedy for fevers and is known to possess the tonic attributes⁸. It is also administered to raise the seminal fluid's viscosity⁹.

It has been used extensively to treat fever, discomfort, and sexual dysfunction¹⁰. The whole plant is used in conventional healthcare to treat a range of illnesses, especially pain, tumors, pile fever, and emollient¹¹. Additionally, there are plenty of traditional therapeutic applications of plant decoction, including for the alleviation of dyspepsia, liver problems, and diarrhea¹².

Inspired by whole plant traditional medical uses, we conducted the phytochemical investigation of the leaves of Corchorus depressus (Linn.). The antibacterial properties and preliminary phytochemical screening of Corchorus depressus (Linn.) leaves using various solvents were documented in our most recent work. Here, we describe the structural characterization and isolation of molecules (PEE-1) and (PEE-2) in an attempt to discover potential bioactive chemicals. The physical and spectral data analysis, along with a comparison with those from previous studies, shed light on the compound’s structures.

MATERIALS AND METHODS:

Plant collection:

Corchorus depressus (Linn.) leaves were collected in Tehsil Sakri, Dhule District (MS), India. The plant specimen was authenticated by Dr. S. R. Kshirsagar, Taxonomist, S.S.V.P.S’s Late Karmaveer Dr. P. R. Ghogrey Science College, Dhule (M.S), India.

Extraction and Isolation of compounds:

Following a thorough washing under running tap water, the fresh plant material's leaves were allowed to air dry. The dried leaves were crushed to a fine powder before being kept in a sealed container. 3kg dried leaf powder was subjected to extraction using petroleum ether solvent by cold maceration¹³, evaporated on a rotary evaporator, and found a dried 4 gm. extract.

A small quantity of petroleum ether extract was dissolved in solvent petroleum ether. The resultant solution was spotted on TLC plates and run by various solvent systems. TLC plate was observed in the iodine chamber and with an Anisaldehyde-H₂SO₄ spraying agent. Following many pilot tests, the components of petroleum ether extracts were separated in an 8:2 solvent system comprising petroleum ether and ethyl acetate. Using column chromatography, 4 grams of petroleum ether extract was gradually eluted on 60-120 grain silica gel with petroleum ether: ethyl acetate solvent systems.

PTLC was carried out to separate two fractions that showed single spots with clear resolution, and the isolated phytoconstituents were called PEE-1 and PEE-2, respectively.

Test for Triterpenoids:

Salkowski’s Test:

A few crystals of PEE-1 & PEE-2 recovered from petroleum ether were placed in different test tubes, to which a few drops of concentrated H₂SO₄ and two milliliters of chloroform were added. The solution has been thoroughly shaken. When two layers were separated, a reddish-brown precipitate appeared at their interface, suggesting that triterpenoids were present in compounds PEE-1 and PEE-2¹⁴.

RESULTS:

Compound Characterization:

PEE-1:

White crystalline solid, m.p. 197-200 ⁰C.

Elemental %: Found C= 84.266 %, H = 11.733 %, O= 4.001 %.

Formula (calculated): C₃₀H₅₀O, Mol. Weight: 426.60.

MS m/z. %: 427 (M⁺).

IR: 3434 (OH-str.), 2978 (aliphatic C-H str.), 1637(C=C str.), 1449-1373 (-CH₂-)

1028-875 (cycloalkanes).

¹H NMR (400MHz, CDCl₃) δ: 5.7 (1H, brs, OH-3), 5.21 (1H. s, H-12), 1.95 (1H, m, H-9), 1.91 (1H, t, H-18), 1.84 (2H, dd, H-11), 1.66 (2H, t, H-21), 1.59 (1H, d, H-19), 1.55 (2H, m, H-2), 1.51 (1H, m, H-1), 1.49 (2H, m, H-6), 1.27 (CH₃-24), 1.22(CH₃-27), 1.19 (CH₃-26), 1.11(CH₃-25), 1.01(CH₃-23), 0.96(CH₃-29), 0.96 (CH₃-30), 0.81(CH₃-28), 0.78 (1H, m, H-5).

¹³C-NMR: (100MHz, CDCl₃) ppm: 145.90 (C-13), 122.03 (C-12), 79.25 (C-3), 55.40 (C-5), 47.89 (C-14), 47.49 (C-19), 47.09 (C-9), 41.93 (C-18), 40.45 (C-8), 40.01 (C-10), 38.80 (C-4), 37.67(C-1), 36.80 (C-21), 34.92(C-22), 33.55 (C-7), 32.86 (C-28), 32.86 (C-17), 32.74 (C-29), 31.30 (C-20), 28.60 (C-27), 28.32 (C-15), 27.49 (C-2), 27.14 (C-23), 26.38 (C-30), 23.71 (C-11), 18.38(C-6), 18.36 (C-16), 17.60 (C-26), 15.89 (C-24), 15.75 (C-25).

PEE-2:

Off-white crystalline solid, m.p. 146-149 ⁰C.

Elemental %: Found C= 76.652 %, H = 9.752 %, O= 13.596 %. Formula (calculated): C₃₀H₄₆O₄, Mol. Weight: 470.51.

MS m/z. %: 471 (M⁺).

IR: 3325 (O-H br. Str.), 2974 (aliphatic C-H str.), 1702 (-C=O str. of carboxylic acid),

1664 (unsaturated ketone -C=O str.), 1453 (-CH₂-), 1025 -756 (cycloalkanes).

¹H NMR (400MHz, CDCl₃) δ: 5.7 (1H, s, H-12), 3.27 (1H, d, H-3), 2.35 (1H, s, H-9), 2.17 (1H, t, H-18), 1.98 (2H, t, H-21), 1.82 (2H, t, H-16), 1.62 (2H, t, H-7), 1.62 (2H, t, H-19), 1.61 (2H, M, H-2), 1.42 (2H, m, H-6), 1.41 (2H, t, H-22), 1.39(CH₃-27), 1.19(CH₃-29), 1.13(CH₃-25), 1.12 (CH₃-26), 1.00 (2H, t, H-15), 0.98 (CH₃-24), 0.95 (2H, m, H-1), 0.93 (CH₃-28), 0.63 (1H, t, H-5), 0.63 (CH₃-23).

¹³C-NMR: (100MHz, CDCl₃) ppm: 203.65 (C-11), 181.68 (C-30), 169.71 (C-13), 128.40 (C-12), 78.85 (C-3), 61.78 (C-9), 54.98 (C-5), 48.23 (C-18), 45.46 (C-8), 43.71 (C-20), 43.20 (C-14), 40.87 (C-19), 39.11 (C-4), 39.11 (C-1), 37.72 (C-22), 37.05 (C-10), 32.74(C-7), 31.86 (C-17), 30.90 (C-21), 28.56 (C-28), 28.49(C-29), 28.09 (C-24), 27.14 (C-2), 26.39 (C-16), 23.48 (C-15), 23.41 (C-27), 18.67 (C-26), 17.47 (C-6), 16.39 (C-25),15.65 (C-23).

Table 1. ¹H (400MHz) and ¹³C NMR (100MHz) data for PEE-1 (β-amyrin) and PEE-2 (Glycyrrhetinic acid)

PEE-1			PEE- 2
δ_C	δ_H	Position	δ_C	δ_H	Position
145.90		13	203.65		11
122.03	5.21	12	181.68		30
79.25	5.7	3	169.71		13
55.40	0.78	5	128.40	5.7	12
47.89		14	78.85	3.27	3
47.49	1.59	19	61.78	2.35	9
47.09	1.95	9	54.98	0.63	5
41.93	1.91	18	48.23	2.17	18
40.45		8	45.46		8
40.01		10	43.71		20
38.80		4	43.20		14
37.67	1.51	1	40.87	1.62	19
36.80	1.66	21	39.11		4
34.92		22	39.11	0.95	1
33.55		7	37.72	1.41	22
32.86	0.81	28	37.05		10
32.86		17	32.74	1.62	7
32.74	0.96	29	31.86		17
31.30		20	30.90	1.98	21
28.60	1.22	27	28.56	0.93	28
28.32		15	28.49	1.19	29
27.49	1.55	2	28.09	0.98	24
27.14	1.01	23	27.14	1.61	2
26.38	0.96	30	26.39	1.82	16
23.71	1.84	11	23.48	1.00	15
18.38	1.49	6	23.41	1.39	27
18.36		16	18.67	1.12	26
17.60	1.19	26	17.47	1.42	6
15.89	1.27	24	16.39	1.13	25
15.75	1.11	25	15.65	0.63	23

DISCUSSION:

PEE-1, a white crystalline substance with a melting point of between 197 and 200 ⁰C, was extracted from the petroleum ether fraction. It gave a positive test for alcohol with the inference from Salkowski's reaction. PEE-1's mass peak (m/z-427, M+) and elemental analysis (C=84.266%, H = 11.733%, O=4.01%) revealed its chemical formula to be C₃₀H₅₀O. The computed formula yields six sites of unsaturation, one for the existence of pentacyclic structure and the other for the C-C double bond. PEE-1's IR spectra shows the existence of an alcoholic O-H group (3434 cm^-1 peak).

The bands at 2978 cm^-1 and 1637 cm^-1 reflected the frequencies of aliphatic C-H stretching and C=C stretching, respectively. The olefin proton H-12 was readily apparent in the compound PEE-1's ¹H NMR spectra, integrating for a single proton at the peak at the 5.21 δ position. In addition, the spectrum showed eight methyl singlets. Peaks at 122.03 and 145.90 in the¹³C NMR spectrum were ascribed to the C-12 and C-13 doubly bonded carbons, respectively. The downfield shift at 79.25 δ corresponds to the C-3 position carbon attached to the hydroxy group. The spectra confirmed the existence of 30 carbon atoms. The full assignments of the compound PEE-1 are shown in Table 1.

The compound-PEE-1 is defined as (3β)-olean-12-en-3-ol (β-amyrin) Fig.1, based on the aforementioned spectrum data and assignments of¹³C NMR signals of the nucleus published in the literature.

PEE-2, an off-white crystalline substance with a melting point of 146-149 ⁰C, was obtained from petroleum ether extract. PEE-2's chemical formula was found to be C₃₀H₄₆O₄ based on its mass peak (m/z- 471, M+) and elemental analysis (C = 76.652%, H = 9.752%, O = 13.596%). The determined formula shows eight bond equivalents which are adjusted for the presence of pentacyclic structure and three remainders for one C-C double bond, a keto group, and a carboxylic acid group.

The IR spectra of PEE-2 reveals the existence of an alcoholic O-H group (3325 cm^-1 peak). The peaks at 1702 cm^-1 and 1664 cm^-1 correspond to carboxylic acid -C=O stretching and conjugated carbonyl stretching, respectively. The peak at 1453 cm^-1 referred to the presence of C=C vibration, while absorption at 2974 cm^-1 represents the C-H aliphatic stretching. ¹H NMR spectra of compound PEE-2 showed a sharp singlet at 5.7 δ representing the olefinic proton H-12. The broad singlet at 6.2 δ represents the carboxylic acid O-H proton. The doublet at 3.27 δ indicates the alcoholic C-3 O-H group. Furthermore, the presence of seven singlets in the spectrum indicated the existence of seven methyl groups. The double bond's carbons C-12 and C-13 are identified by the peaks in the molecule PEE-2's ¹³C NMR spectra, which are located at 128.40 δ and 169.71 δ. The peaks at 203.65 δ and 181.68 δ evident the presence of conjugated carbonyl and carboxylic acid groups respectively. Table 1 lists all of the chemical PEE-2's assignments. Based on spectrum data and assignments of ¹³C NMR signals of the nucleus found in the literature, the compound-PEE-2 is identified as (3β)-hydroxy-11-oxoolean-12-en-30-oic acid (Glycyrrhetinic acid) Fig.1.

β-amyrin (PEE-1)

Glycyrrhetinic acid (PEE-2)

Fig.2 Isolated compounds from Corchorus depressus (Linn.)

CONCLUSION:

Two known compounds, PEE-1 and PEE-2, have been identified and isolated as a result of the current investigation on the leaves of the plant Corchorus depressus (Linn.). The structures of the compounds were identified using elemental analysis, spectral analysis, and a comparison of the literature. The compounds were identified as β-amyrin and Glycyrrhetinic acid

CONFLICT OF INTEREST:

The authors declares no conflict of interest.

LIST OF SYMBOLS AND ABBREVIATIONS:

Table 2: Abbreviations

Abbreviations	Meaning
PEE-1	Petroleum Ether Extract-1
PEE-2	Petroleum Ether Extract-2
PTLC	Preparative Thin Layer Chromatography
MS	Mass Spectroscopy
IR	Infrared Spectroscopy
¹H NMR	Hydrogen Nuclear Magnetic Resonance Spectroscopy
¹³C NMR	Carbon Nuclear Magnetic Resonance Spectroscopy
δ_C	Chemical Shift (in δ) of Carbon atom
δ_H	Chemical Shift (in δ) of Hydrogen atom

ACKNOWLEDGEMENTS:

The authors acknowledge the Principal of Sandip Institute of Technology and Research Centre, Nashik, for providing the essential facilities. This work was also supported by the Principal, P.S.G.V.P. Mandal's S.I.Patil Arts, G.B.Patel Science, and S.T.K.V.S Commerce College, Shahada, District-Nandurbar, who provided the research facilities.

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Received on 02.01.2024 Modified on 07.03.2024

Research J. Pharm. and Tech 2024; 17(10):4667-4670.

DOI: 10.52711/0974-360X.2024.00719