PHYTOCHEMICAL ANALYSIS OF CHLOROFORM EXTRACT OF ROOTS OF KALANCHOE PINNATA BY HPLC AND GCMS

PHYTOCHEMICAL ANALYSIS OF CHLOROFORM EXTRACT OF ROOTS OF KALANCHOE PINNATA BY HPLC AND GCMS

Quazi Majaz*, Sayyed Nazim, Siraj Shaikh, Pravin Gomase and Amol Choudhari

Ali-Allana College of pharmacy, Akkalkuwa, Dist- Nandurbar, Maharashtra, India

ABSTRACT

The plant Kalanchoe pinnata is widely used in ayurvedic system of medicine as astringent, analgesic, carminative and also useful in diarrhea and vomiting. Naturalized throughout the hot and moist parts of India. And lots of phytochemical and pharmacological work has done on leaves of plant but the root part is not focused. Hence we have selected roots for phytochemical analysis which will support further evaluation of pharmacological activity. In this first roots are subjected to chloroform for extraction. And preliminary phytochemical test were performed. Chloroform extract was subjected for HPLC and GC-MS analysis which shows various phytoconstituents present in extract.

GC-MS

INTRODUCTION: Various species of KALANCHOE are used medicinally in Indo-China and Philippines Islands, whereas Kalanchoe pinnata Pers. (Family Crassulaceae) is naturalized throughout the hot and moist parts of India. The leaves and bark is bitter tonic, astringent to the bowels, analgesic, carminative, useful in diarrhoea and vomiting ¹. Antiulcer ², anti-inflammatory ^3-4 and antimicrobial activity ⁵ of leaf extract was reported.

Oral treatment with leaf extract significantly delayed onset of disease in BALB/c mice infected with Leishmania amazonensis as compared to untreated mice or mice receiving K. pinnata by the intravenous or topical routes ⁶. Potent cytotoxic compounds bersaldegenin- 1, 3, 5-orthoacetate ⁷ and bufadien- olide-bryophyllin B ⁸ were isolated. Other chemical constituents from this plant are bryophyllol, bryophollone, bryophollenone, bryophynol and two homologous phenanthrene derivatives 2(9-decenyl)-phenanthrene (I) and 2-(undecenyl)-phenanthrene (II) from leaves ⁹. Isolation and structure elucidation of 24-epiclerosterol [24(R)-stigmasta-5, 25-dien-3β-ol], 24(R)-5α-stigmasta-7, 25-dien-3β-ol, 5α-stigmast-24-en-3β-ol and 25-methyl-5α-ergost-24 (28)-en-3β-ol from aerial parts was done ¹⁰.

This species is also included in the plants species, which are used by the tribals of Kerala for treating cancer symptoms ¹¹. Juice of the fresh leaves is used very effectively for the treatment of jaundice in folk medicines of Bundelkhand region of India, but no systemic study to assess this activity has been carried out.

As the aerial parts of plant have many pharmacological activity but roots of this plant was not focused yet hence the present investigations were carried out to evaluate phytoconstituent of roots of plant with the help of HPLC and  GC-MS, which will help for further pharmacological evaluation.

MATERIALS AND METHODS:

Collection of Plant Material: The roots of Kalanchoe pinnata was collected from Satpuda hills near Akkalkuwa, Dist: Nandurbar, Maharashtra, India, in June 2010, cleaned and dried at room temperature in shade and away from direct sunlight. The plant authenticated by T. Chakraborthy, Deputy Director, Botanical Survey of India, Koregaon Road Pune, by comparing morphological features and a sample voucher specimen of plant was deposited for future reference (Voucher specimen number QMAKP1).

Preparation of Extract: The root of Kalanchoe pinnata was collected and dried in the shade and then pulverized in a grinder. The powdered drug was utilized for extraction. Material was passed through 120 meshes to remove fine powders and coarse powder was used for extraction. A method described in Mukherjee was used for extraction of powdered plant. Extraction was done by Chloroform ¹².

Preliminary Phytochemical Screening: The extract was then subjected to preliminary phytochemical screening to detect the presence of various phytoconstituent. The results show presence of Steroids, Saponins, Alkaloids, Glycosides, Flavonoids, Tannins in the chloroform extract ¹³.

HPLC Analysis for Flavonoids ¹⁴:

• Column: Hypersil- ODS Column (250mm × 4.6mm), 5µm particle size
• Mobile phase: Acetonitrile: Phosphate buffer pH 2.4 (25:75)
• Flow rate: 1.2 ml/ min
• Detection: 266 nm

HPLC analysis for steroids ¹⁵:

• Column: Hypersil- ODS Column (250mm × 4.6mm), 5µm particle size
• Mobile phase: Methanol: Water (95:5)
• Flow rate: 1ml/ min
• Detection: 210 nm

GC-MS Analysis ¹⁶: The GC-MS analyses were carried out in Perkin Elmer, Auto system XL GC+.

• Carrier gas: helium with a flow rate of 0.7 mL/min;
• Column temperature: 5 min in 180°C, 180-260°C at 3°C/min, 5 min in 260°C, 260-280°C at 0.2°C/min, and finally 5 min in 280°C;     injector temperature, 280°C detector temperature, 290°C.
• Volume injected: 1 µL of sample
• Ionization potential: 70 eV
• Ion source temperature: 290°C.

RESULTS AND DISCUSSION:

HPLC Analysis for Flavonoids: Analysis of flavonoids in chloroform extract of plant shows 15 compounds (Graph 1) after comparing with standard retention time of various flavonoids, it shows peak at 7.4000 which is nearer to myrcetin ¹⁴.

GRAPH 1: CHROMATOGRAM OF HPLC FOR CHLOROFORM EXTRACT FOR FLAVONOIDS

TABLE 1: RESULTS OF HPLC FOR CHLOROFORM EXTRACT FOR FLAVONOIDS

HPLC Analysis for Steroids: Analysis of flavonoids in chloroform extract of plant shows 8 compounds (Graph 2) after comparing with standard retention time of various flavonoids, it shows peak at 7.8920 and 10.1563 which is nearer to cholesterol and beta sitosterol respectively ¹⁵.

GRAPH 2: CHROMATOGRAM OF HPLC FOR CHLOROFORM EXTRACT FOR STEROIDS

TABLE 2: RESULTS OF HPLC FOR CHLOROFORM EXTRACT FOR STEROIDS

GC-MS Analysis: After preliminary phytochemical investigation and HPLC analysis shows the presence of various phytoconstituent in chloroform extract (Graph 3). Further investigation done by GCMS. Gaschromatograph of chloroform extract shows the presence of 40 phtoconstituent (Table 3) which are subjected to mass spectroscopy. The mass spectroscopy of these peaks does not give any prominence result it shows only presence of steroids and various sugar molecule (Graph 4), here ms data of  chloroform extract for peak at 37.075 RT (Table 4). The list of possible phytoconstituent is given by checking compound in NIST and NBS library.

GRAPH 3: GC CHROMATOGRAM OF CHLOROFORM EXTRACT

TABLE 3: AREA PERCENT REPORT OF CHLOROFORM EXTRACT

GRAPH 4: MS DATA FOR CHLOROFORM EXTRACT FOR PEAK AT 37.075 RT

TABLE 4: LIST OF PROBABLE COMPOUND OF CHLOROFORM EXTRACT FOR PEAK AT 37.075 RT

 CONCLUSION: Preliminary phytochemical screening of chloroform extracts of K.Pinnata shows the presence of Steroids, Saponins, Alkaloids, Glycosides, Flavonoids and Tannins. HPLC analysis shows the presence of myrcetin, cholesterol and beta sitosterol. While but GCMS data show only steroids and sugar moieties, no molecule from flavonoids. The possible reason should be due lack of derivetisation technique which degrade the flavonids on heating into glycon and aglycon moiety.   From these above phytochemical investigation it was concluded that chloform extract of Kalanchoe pinnata root contain various flavonoids and steroids. Which will leads further pharmacological investigation of this plant.

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