Exploration of Phytopharmacognostical Study of Ipomoea obscura (Linn.) Ker Gawl

indole alkaloids


INTRODUCTION
Ipomoea obscura (L.), commonly described to this in Ayurveda as "Laksmana, " is an herb in the Convolvulaceae family. It's a little climber with small cordate leaves with apex that's acuminate. Five fully united petals make up the corolla. In disturbed environments, the plant grows on fences or as a low ground cover as a substrate. It's a slender, twinning perennial herb that grows in grasslands, hedges, and wastelands all over India, up to a height of 3000 feet. As a climber with lovely flowers, it also has decorative value. [1] This plant's medicinal benefits have been found by Ayurveda, and it is used to treat diarrhoea, open sores, and pustules. Ulcers, haemorrhoids, and swellings are treated with a leaf paste. Fruits and seeds are being used as purifying agents, to help with difficult breathing, to relieve pain, and also to improve vision. [2] To treat colds, asthma, and dry cough, gingely oil is combined with the fresh plant extract.By widening blood vessels, the plant extract relieves pain in the body. Many siddha medications contain plant extracts. [3] Other phytochemicals found in this plant were flavonoids, steroids, essential oil, and phenolic acids. [4] This plant contains indole alkaloids like Ipobscurine A, Ipobscurine B, Ipobscurine C, and Ipobscurine D as well as tropane alkaloid including Calysteginine B-1,2, 3 and 4. Calysteginine C-1; [5][6] Ipobscurine-A, C, and D are significant pharmacologically active macrolactum type indole alkaloids found in Ipomoea obscura.
The anti-inflammatory and anti-angiogenic effects of these indole alkaloids have been documented. [7] Bulnesene (23.8%), -humulene (13.7%), and seychellene were the three primary components of the essential oil (11.2 percent). Other trace amounts of -guaiene (8.3%), -caryophyllene (7.1%), -terpinene (4.2%), -hexadecanoic acid (3.0%), and -elemene were present (2.7 percent). The oil was found to have many components of the sesquiterpene hydrocarbon class (78.4%). [8] Ipomoea obscura extracts have been shown to exhibit anti-inflammatory, nephroprotective, anti-angiogenic, and immunological modulatory properties. [9] Ipomoea obscura whole plant extracts have high antioxidant activity against a variety of free radicals. [10] Ipomoea obscura methanol extracts have been demonstrated to have good cytotoxic and moderate antibacterial properties. [11] The literature review revealed that the separation and characterization of phytoconstituents from the plant had not been investigated, thus the investigations were carried out in the current study.

Plant Material Authentication and Collection
Ipomoea obscura leaves were collected from several locations in the Nashik region in Maharashtra, India.
Standard literature was used to confirm plant species identification. Dr.Shimpi, Taxonomist, Head of Botany Department, G. E. Society's NSC Science College, Nashik, India, validated the plant sample. The voucher specimen No. 508 for Ipomoea obscura (L.) Ker Gawl was preserved in the college herbarium (NSCSC/PCG/2017/12) for future reference. To remove any dust, the leaves were rinsed with tap water and then dried in the shade. With the use of a grinder mixture, the dried leaves material was ground into a fine powder. The powdered leaves were kept in an airtight container for further usage.

Microscopical Examination
The exterior morphology was investigated using the methods described in the literature. A basic microscope was used to conduct the macroscopic research. Leaves, flowers, and seeds were studied for colour, odour, taste, size, and morphology. [12] Microscopic Analysis Thin leaf fragments were prepared for microscopic examination. The thin slices were washed in water and dyed with safranin, haematoxylin, picric acid, and dil. liquid iodine before mounting in glycerine for clarity and confirmation of magnifications. (10X, 45X). [13][14]

Physicochemical Investigation
It is impossible to overestimate the relevance of physicochemical standards in establishing the quality, purity, and adulteration of a given crude medicine. The amount of care used in collecting and preparing a medicine for market, as well as the amount of foreign matter in a natural drug, are reflected in the ash values. Traces of organic matter that ordinarily interfere with analytical determination were removed by determining the ash value. In general, ash from incineration comprises phosphates and carbonates, as well as calcium, potassium, sodium, and magnesium in the form of silicates. The extractive value, moisture content, and total, acid-insoluble, water soluble and sulphated ash values were assessed using Indian Pharmacopoeial techniques. [15][16] Methodology of Extraction 250 g of powdered plant material was packaged in white cotton bags and extracted in a Soxhlet device using several solvents in a sequential manner. Petroleum ether (60-80°C), chloroform, and methanol were utilised as extraction solvents. In an oven below 50°C, the powdered material was dried before extracting with the next solvent. These consecutive extracts were subjected to extractive values and preliminary phytochemical analyses. [17]

Phytoconstituents: A Qualitative and Quantitative Analysis
To confirm the existence of phytoconstituents, protein, alkaloids, phenolic compound, terpenoid, glycosides, phytosterols and flavonoids and other substances, preliminary phytochemical analyses on successive extracts were carried out in this work. The approach was also used to calculate the total phenolic and flavonoid content of the extracts. Total phenol and total flavonoid concentrations were calculated in milligrams of gallic acid equivalents per gram of extract and miligrames of rutin equivalents per gram of extract, respectively, using the mean of three readings. [15] Isolation and Characterization of Phytoconstituents from Ipomoea obscura Leaf; [18][19] Before being extracted (1 kg) in a Soxhlet extractor for 18-20 hr with 4.5 L methanol, the fresh whole plant was dried in the shade, ground up, and sieved No. 20. The extracts were dried using a rotary evaporator at a controlled temperature between 40-50°C and lower pressure. It dried to a 655 g solid residue that was dark yellowish green. The unsaponifiable material was isolated using the n-hexane fraction after the methanolic extract was partitioned in n-hexane and water for 3 hr (240 g). The aqueous layer then treated three times (100 mL) with carbon tetrachloride that before bottom layer of carbon mono tetrachloride was removed and concentrated (35 g) (80 g).

Isolation and Characterisation of Flavonoids from I. obscura
After partitioning the crude MIO extract, the n-hexane fraction (45.5 g), dichloromethane fraction (25.4 g), and Ethyl acetate fraction (48.7 g) were obtained. To get 15 fractions, the ethyl acetate fraction (40.0 g) was separated using silica column chromatography eluting with mixes of n-hexane/Ethyl acetate (100:0-0:100 v/v), Ethyl acetate/ Methanol(100:0-0:100 v/v), and Ethyl acetate/Methanol (100:0-0:100 v/v) (Fr. A to Fr. O). Fraction D (14.1 g) was separated into 5 sub-fractions (D1-D5) on a silica gel CC using a 9:1 v/v combination of n-hexane and Ethyl acetate. CC split fraction D4 (1.1 g) into 3 sub-fractions (D4.1 to D4.3) using Ethyl acetate. Recrystallization purified fraction D4.1 (361.2 mg) to get compound IO3 (211.7 mg). CC purified fraction D4. 2 (188.9 mg) by using Chloroform/Methanol (5:1 v/v) to produce compound IO4 (17.4 mg). The thin layer chromatography (TLC) was used to examine all of the obtained fractions, and the fractions with identical Rf values were merged. Recrystallization was used to purify the principal portions. Spectroscopic methods (UV, FTIR, and NMR spectroscopy) were used to characterise all of the isolated chemicals. [20][21][22] HPTLC fingerprinting analysis; [23][24][25] The research's main objective was to evaluate and enhance the HPTLC fingerprint technique for standardization crude drugs. The sample was applied with a microlitre syringe using a Camaglinomat IV applicator with a band width of 7 mm to precoated silica gel plates 60 F 254 [10 cm × 10 cm, 0.2 mm thickness, E. Merck]. The plates were created in a solvent system and then saturated with the solvent for 20 min in a Camag twin through glass chamber. They were separated by 8 centimetres. After scanning on a Camag TLC Scanner in absorbance at 254, 365, and 740 nm with wincats software 4.03, TLC plates were air dried. The HPTLC profile were summarised in Table 1

Macroscopic Characteristics
It's a little climbing vine with alternately arranged short cordate leaves with whole margins and acuminate apex. Flowers are solitary or in cymes, with peduncles as long as the petioles. The corolla is made up of five fully united white petals with a scarlet core. The corolla tube's neck has five stamens joined to it. With two locules and numerous seeds, the ovary is superior. At maturity, the fruit is a sub-globose, glabrous capsule.

Microscopic Study
Leaf microscopy: Transverse section of the epidermis reveals uniformity, with somewhat larger cells on the dorsal side due to noticeably higher anticlinal walls. The paracytic stomata are on the same level as the neighbouring cells, and the guard-cells have discernible outer cuticular ledges. About half of the height of the chlorenchyma is made up of the dorsiventral region of the mesophyll, which is comprised of two or three layers of palisade parenchyma and four to six rows of spongy parenchyma. There were discovered to be 2 -3 layers on the dorsal side and 8 to 10 layers on the adaxial side of the angular collenchyma that makes up the epidermis. Several ovate, ring-shaped vascular bundles that was ancillary with fascicular cambium and cross the ground parenchyma. Four cells with thick cytoplasm, a visible nucleus, and the ability to secrete lipophilic substances are found in a number of secretory ducts with uniseriate epithelium that are frequently aligned by the arterial bundles (Figure 1 A) Microscopy of the stem: The epidermis of juvenile branches is thicker, with sclerotized outer cell walls that are robust and hairy, like a piece of glands, as in the leaf. Collenchyma is a big clump of cells found beneath the epidermis. Cells of the epidermis with thin beaded walls, stomata are tiny, round, and have a prominent stomatal slit bordered by 2-4 paracytic cells. Mucilage inclusions are found in epidermal stem cells. The epidermis has a thick layer of hair on it. Pith occupies a substantial percentage of the stem's central region, while hypodermis exists outside of it. Pith cells in the shape of barrels store a lot of food. Vascular bundles are arranged in rays that are parallel to each other (Figure 1 B).

Methodology of Extraction
The extractive value was determined to be highest when extraction was done with methanol, chloroform, and lowest when extraction was done with petroleum ether, i.e. 15.25 %, 9.34 %, and 5.82 %, respectively. Table 5 shows the results. In Ipomoea obscura, maximal extraction with methanol has also been recorded.

Quantitative and qualitative investigation of phytoconstituents
The qualitative phytochemical analysis of the plant extract as shown Table 6 revealed the presence of steroids, saponins, alkaloids, glycosides,      5.56 percent (Table 7 and Figure 2). The total concentration of flavonoids was determined using the calibration. The total flavonoid content of the methanolic extract of I. obscura leaves was examined using an aluminium chloride colorimetric method and a calibration curve of rutin at various concentrations, and it was discovered to be 0.34 percent (Table 8 and Figure 3).

Isolation and Characterization of I. obscura Leaves
Isolation and characterization of terpenoid from I. obscura leaves Compound IO1: The Lieberman-Burchard steroid test was passed by compound IO1. Crystals of compound IO1 with a melting point of 214-215 degrees Celsius were isolated as the substance. The IR spectra revealed the presence of such -OH group at 3303 cm -1 , C-O at 1732 cm -1 , and 1637 cm -1 (Figure 4). The 1H NMR for -CH3 protons showed signals at (ppm) 0.83, 1.02, 0.79, and 1.70. The carbonyl proton was discovered as a broad singlet at 5.11, whereas the olefinic resonance was discovered as a singlet at 4.56. In Figure 5, A comparison of the spectroscopy data from compound IO1 with the literature led to the discovery that it is lupeol.
Compound IO2: The Lieberman-Burchard steroid test was passed by compound IO2. With a melting point of 147°C, the compound IO2 was isolated as colourless crystals. The IR spectrum confirmed the existence of the hydroxyl group at 3433.06 cm -1 and alkene (C=C) at 1639 cm -1 Figure 4: FI-IR of compound IO1.   ( Figure 6). The 1H NMR (Figure 7) showed indications for methyl protons at 1.25, 1.01, 0.88, and 0.93. The carbonyl proton was discovered as a broad singlet at 5.01, whereas the olefin resonance was discovered as a multiplet at 3.52. The 13C NMR peaks at 140.09 and 121.4 proved that an olefin bond existed between C-5 and C-6 (Figure8). Following a comparison of the produced spectrum data with published data, compound IO2 was determined to be a β-sitosterol.
Isolation and characterization of flavonoids from I. obscura leaves Compound IO3: Compound IO3 was created as an amorphous, yellow powder. Two sizable peaks of absorption at 265 and 365 nm were visible in the compound's UV spectra. These absorption peaks are typical of flavonols, which also exhibit significant peaks in the 200-400 nm range. Band I (between 300 and 380 nm) and band II (between 400 and 450 nm) refer to these two peaks (240-280 nm). Band I deals with the B-(the ring's cinnamoyl system's) absorption, while Band II deals with the A-(the ring's ring's) absorption (benzoyl system). The presence of hydroxyl and carbonyl functional groups was detected at 3253 and 1651 cm -1 , C-H stretching at 2956 cm -1 , C=C olefin ring at 1361 cm -1 , asymmetric C-O-C stretching at 1171 cm -1 , and substituted benzene at 828 cm -1 , according to IR measurements ( Figure 9). The 1H NMR spectrum of 1 revealed four aromatic hydrogen signals comprising six protons ( Figure 10)     molecule. There were 19 carbon signals seen in the 13C NMR spectra ( Figure 11). H-6 (6.24 ppm)/C-6 (98.5 ppm), H-8 (6.45 ppm)/C-8 (92.5 ppm), H-2 or H-6 (7.85 ppm)/C-2 and or C-6 (131.4 ppm), and H-3 or H-5 (7.05 ppm)/C-3 and or C-5 were the six methines that were found to be aromatic in IO3 (115.1 ppm). We identified this isolated substance IO3 as kaempferol-3-O-rhamnoside by comparing the spectrum data of the isolated compound with published research.
Compound IO4: Compound IO4 was isolated as a yellow amorphous powder. Two distinct high absorption peaks at 256 and 372 nm were visible in the compound IO4's UV spectra. IR absorption bands at 3296 cm -1 , 1659 cm -1 , 1596 cm -1 , and 1169 cm -1 , respectively, confirmed the presence of hydroxyl, carbonyl, aromatic ring, and ether groups ( Figure 12). In Figure 13, the molecule IO4's 1 H-and 13 C-NMR spectra, aromatic systems showed resonances. The presence of hydroxyl groups at C-4, C-6, and C-3 and C-4 of the B ring corresponds to the existence of a strong singlet signal at 4. 87 in the 1 H-NMR spectrums ( Figure 15). Another broad singlet is seen at position 12. This may indicate that OH is present at the C-3 position next the carboxylic group. The existence of 15 aromatic carbon signals could be seen in the 13C NMR spectra ( Figure 14). The presence of carbonyl carbon was suggested by the signal at 179. The structure of compound IO4 was predominantly identified as a Quercetin based on the obtained spectral data and comparison with the data provided in the literature.

HPTLC Fingerprinting
HPTLC fingerprinting of I. obscura with lupeol The resolution of the chosen mobile phase was excellent. HPTLC chromatogram of standard lupeol revealed a single peak (Figure 15 and 16). After the chromatogram was developed, the plate was derivatized with vanillin-H 2 SO 4 , which produced a violet colour spot in both the standard and the n-hexane fraction of IO. The plate was then scanned at 540 nanometers. The R f value of the chemical found in the n-hexane fraction of IO (0.52) was virtually identical to that of the standard (0.53) ( Table  9). When the R f values of the IO extract are compared to the standard, HPTLC finger printing confirms the presence of Lupeol.

HPTLC fingerprinting of CCl4 extract with β-sitosterol
The resolution of the chosen mobile phase was excellent. HPTLC chromatogram of standard -sitosterol revealed a single peak (Figure 17 and 18). After the chromatogram was developed, the plate was derivatized with the visualizing agent vanillin-H 2 SO 4 , which revealed a pink colour spot in both the standard and extract. The plate was then scanned at 540 nanometers. The Rf value of the chemical found in the CCl4 fraction of IO (0.39) was almost identical to that of the standard (0.39). (Table 10). When the Rf values of the CCl4 fraction are compared to the standard, it indicates the existence of β -sitosterol in the IO extract.    (Table 11), and fluorescence indicated the presence of quercetin and kaempferol.

CONCLUSION
The Ipomoea obscura extract contains a variety of phytochemicals such as reducing sugars, glycosides, phenolic compounds, flavonoids, steroids, and alkaloids, according to the phytochemical study. The quantitative analysis showed the presence of 5.56% phenolic component and 0.34 % flavonoids. The phytochemical investigation of Ipomoea obscura led to the discovery of triterpenoid and flavonoid components. These chemicals were purified by crystallisation after being isolated using various scientific procedures. The chemical structure of lupeol and β-sitosterol in n-hexane fraction, quercetin and kaempferol in carbon tetrachloride fraction of methanolic extract of Ipomoea obscura was determined using UV-Vis, IR, 1 H, and 13 C NMR spectroscopic techniques. The abovementioned study provides information on the identification of I. obsura, chemical components, and physical features. These studies assist in the identification and verification of plant material.

HPTLC fingerprinting of fraction D of IO with standard Quercetin and Kaempferol
Various solvent compositions with varying polarities were used to optimise the mobile phase. In the fraction D of the mobile phase Tolune: