Phytochemical Analysis of Flower extracts of Different Cassia Species by Using Gas Chromatography-Mass Spectrometry

MATERIALS AND METHODS

Plant materials: The flowers of C. alata, C. auriculata, C. fistula and C. occidentalis were collected from Thanjavur District, Tamil Nadu, India from January-April, 2018. About 1.0 kg of shade-dried coarse powders of the flower material was extracted with 80% v/v aqueous ethanol by maceration at room temperature for 72 h. After the completion of each extraction, the extracts were filtered, concentrated to dryness in a rotavapor under reduced pressure and controlled temperature (40-50°C). The residues were stored in a vacuum desiccator for further use.

GCMS: The ethanolic flower extracts were subjected to GC-MS analysis on the instrument GC and MS JEOL GC mate equipped with secondary electron multiplier. The JEOL GC MATE II GC-MS with data system is a high resolution, double focusing instrument. Maximum resolution: 6000 Maximum calibrated mass: 1500 Daltons. Source options: Electron impact (EI); Chemical ionization (CI) (Agilent Technologies 6890N Network GC system for gas chromatography). The column (HP5) was fused silica 50 m×0.25 mm I.D. Analysis conditions were 20 min at 100°C, 3 min at 235°C for column temperature, 240°C for injector temperature, helium was the carrier gas and split ratio was 5:4. The sample (1 μL) was evaporated in a split less injector at 300°C. Run time was 22 min.

Chemical composition by GCMS analysis: Interpretation on mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST). The spectrum of the unknown components was compared with the spectrum of the known components stored in the NIST library.

RESULTS

GCMS analysis of C. alata flowers: The chromatogram (Fig. 1) shows 12 prominent peaks. The largest peak (RT 18.55) is due to the presence of Oleic acid. From the GCMS study, 12 phytocompounds were identified from C. alata flowers, shown in Table 1.

GCMS analysis of C. auriculata flowers: The chromatogram (Fig. 2) shows 9 prominent peaks. The largest peak (RT 16.55) is due to the presence of n-Hexadecanoic acid. The Second less prominent peak ( RT 18.13) is due to the presence of Oleic acid. Flavone also found with less intense peak (RT 14.85). The identified phyto constituents from C. auriculata flowers were shown in Table 2.

GCMS analysis of C. fistula flowers: The GC-MS analysis of C. fistula flowers showed the largest peak (RT 19.2) is due to the presence of Octadec-9-enoic acid, classified as a member of the Long-chain fatty acids (Fig. 3). The identified components are shown in Table 3.

GCMS analysis of C. occidentalis flowers: Chromatogram shows 13 phytocompounds identified from the C. occidentalis flower extract (Fig. 4). The largest peak (RT 18.6) is due to the presence of Oleic acid. Oleic acid is classified as a monounsaturated omega-9 fatty acid. The identified components are shown in Table 4.

DISCUSSION

In the present research, an attempt has been made to identify phytochemical studies in flowers of some Cassia sp. namely C. alata, C. auriculata, C. fistula and C. occidentalis by GCMS. In the previous study, 3 species of Cassia namely Cassia alata Linn., Cassia occidentalis Linn. and Cassia sieberiana DC were carried out to identify and quantify the bioactive components of these ornamental shrubs. The alkaloid, flavonoid, saponin, steroid, phenol and tannin contents of the vegetative and reproductive parts of these plants viz. the leaves, stems and roots were screened and compared while the flowers were not analyzed¹⁹.

Fig. 1:	Chromatogram of Cassia alata flowers

Fig. 2:	Chromatogram of Cassia auriculata flowers

Fig. 3:	Chromatogram of Cassia fistula flowers

Fig. 4:	Chromatogram of flowers of Cassia occidentalis

Deshpande and Bhalsing¹⁹ presented a review of the phytochemistry of some medicinally important Cassia sp., because there are about 580 species of this genus “Cassia” scattered all around the world. Only 46 species have been phytochemically studied. Most of the studies carried out with leaves, barks and stems²⁰. In the present study, flower extracts were used and compared, most of the identified compounds were hydrocarbons, fatty acids, fatty esters, flavones and coumarin derivatives. Many of these identified compounds have already been reported to be pharmacologically active. For example, hexadecanoic acid is known to have potential anti-bacterial and anti-fungal activities, long-chain unsaturated fatty acids, such as oleic acid, also show anti-bacterial activity and are the key ingredients of anti-microbial food additives and some anti-bacterial herbs²¹, unsaturated fatty acids are also suggested to be responsible for the anti-inflammatory activity²² and hexadecanoic acid, methyl ester, phytol have shown anti-oxidant and anti-cancer properties, respectively^23,24. Oleic acid identified as the major phytocompound in C. alata, C. auriculata and C. occidentalis. The principal use of oleic acid is as a component in many foods, in the form of its triglycerides. It is a component of the normal human diet as a part of animal fats and vegetable oils. Oleic acid as its sodium salt is a major component of soap as an emulsifying agent and it is used as an emulsifying or solubilising agent in aerosol products²⁵. Oleic acid also found in Daniellia oliveri stem bark extract²⁵. Flavone found in C. auriculata and C. Occidentalis flowers. Flavones, are a class of flavonoids based on the backbone of 2-phenylchromen-4-one (2-phenyl-1-benzopyran-4-one). Flavones represent one of the largest groups and possess a functional C2=3 double bond in the basic structure²⁶. Flavones are important anti-oxidants and promote several health effects. Aside from anti-oxidant activity, these molecules provide the following beneficial effects: Anti-viral, anti-cancer, anti-inflammatory, anti-allergic²⁷. A steroid, estra-1,3,5(10)-trien-17á-ol present in C. alata and C. occidentalis it may be responsible for the observed estrogenic and/or anti-estrogenic activity²⁸. Pregna-5,8,16-triene-3à-ol-20-one acetate present in C. occidentalis flowers, its chemical name is 16-Dehydropregnenolone acetate. A 16-Dehydropregnenolone acetate is the dehydration product of pregnenolone acetate. 16-Dehydropregnenolone acetate is used in the preparation of pregnane derivatives and its glycosides as potential anticancer agents²⁹. Isopropyl stearate is found in C. fistula flowers. It is used in cosmetics and personal care products, most frequently in the formulation of eye makeup, skin makeup, skin conditioning agent, lipstick and skincare products.

Table 1:	Compounds identified in Cassia alata flowers

Phytol is found in C. fistula flower extract with RT 18.48. Phytol is known to be anti-microbial, anti-cancer, anti-inflammatory, hepatoprotective³⁰. Phytol also found in Amaranthus caudatus³¹, Broussonetia luzonica³². Palmitic acid or n-Hexadecanoic acid was found in C. auriculata flower extract. It is a saturated fatty acid that has anti-androgenic, anti-fibrinolytic, hemolytic 5-alpha reductase inhibitor, antioxidant, hypo-cholesterolemic, anti-inflammatory, phospholipase inhibitor, anti-cancerous, anti-microbial, nematicide and mosquito larvicidal properties^33-38. Recently, n-Hexadecanoic acid was found in different plant parts of Leptadenia reticulate³⁹. Biological activity of some of the identified compounds listed in Table 5. This study summarized phytoconstituents of some genus Cassia, which can be further investigated for the improvement of new novel herbal drugs. Due to medicinal properties there is enormous scope for future research on Cassia sp. and further clinical and pharmacological investigation should be conducted to investigate unexploited potential of this plant.

Table 2:	Compounds identified in Cassia auriculata flowers

Table 3:	Compounds identified in flowers of Cassia fistula

Table 4:	Compounds identified in the flowers of Cassia occidentalis

Table 5:	Biological activity of some identified compounds

CONCLUSION

The Cassia sp. have been studied for their phytoconstituents. Flavone was widely distributed throughout this genus which suggests that these compounds may be chemotaxonomic markers of the genus Cassia. The genus Cassia definitely holds the promise of providing potent drugs for both chronic and acute diseases like diabetes mellitus and others. It is also clear that much needs to be discovered, both as to the active ingredients and their biological effects. The results presented here are intended to serve as a reference tool to researchers in the fields of ethnopharmacology.

SIGNIFICANCE STATEMENT

Over the years, many scientists have performed research on different plant families to be able to identify secondary metabolites. Present study discover many secondary metabolites that can be beneficial for the researchers in the field of ethnopharmacology This paper summarizes GCMS studies on some flowers of Cassia sp., which can be further investigated for the improvement of new novel herbal drugs. The detailed information regarding the phytoconstituents of Cassia sp. can be useful for the development of new traditional medicine and for the benefit of the mankind. This study will help the researchers in identifying active components of Cassia sp. in future. Thus a new theory on it may be arrived at.

ACKNOWLEDGMENT

Authors sincerely thank Sophisticated Analytical Instrumentation Facility (SAIF), IITM, Chennai for GCMS analysis.