PHYTOCHEMICAL ANALYSIS AND ANATOMICAL STUDY OF TWO SPECIES OF CESTRUM FROM CHANDIGARHHTML Full Text
PHYTOCHEMICAL ANALYSIS AND ANATOMICAL STUDY OF TWO SPECIES OF CESTRUM FROM CHANDIGARH
Nishtha*, Richa and Anju Rao
Department of Botany, Panjab University, Chandigarh - 160014, Chandigarh, India.
ABSTRACT: Family Solanaceae is known for its tropane alkaloids which have many medicinal properties. In our study two Cestrum species namely Cestrum diurnum and Cestrum nocturnum which are also ornamental plants are studied as different parts of the plants possess anti-bacterial, anti-malarial, analgesic, anti-inflammatory, inhibitory effect on central nervous system and wound healing properties. The present study was undertaken to analyse the presence of various phytochemicals such as alkaloids, flavonoids, terpenoids, saponins, steroids through preliminary qualitative phytochemical screening of the leaf extract. HPLC(High Performance Liquid Chromatography) was used for the qualitative and quantitative active principle analysis which revealed the presence of nicotine and nornicotine as major alkaloids and comparative study of the two species showed the presence of these alkaloids in large quantity in Cestrum diurnum. The detailed anatomical studies of the part(s) used help in the correct easy identification of the commercial drug in its purest form.
Phytochemicals, Cestrum species, Anatomical studies, HPLC
INTRODUCTION: Plants are the richest source of medicine and have been used since ages for their therapeutic value. A large no. of new pharmacologically active agents which have been explored from natural resources and plants are one of them which lead to the discovery of many clinically useful drugs. The useful plant based drugs contain active principle ingredients which are secondary metabolites also called phytochemicals present in the plants in any part that can be leaves, roots, stem, seeds etc. These phytochemicals can be alkaloids, saponins, tannis, flavonoids and many more.
Two plants namely Cestrum diurnum and Cestrum nocturnum have been selected for the present study from the different areas of Chandigarh. Cestrum diurnum L., (Day Jasmine), also known as “Din ka Raja” blooms during day time is a member of Solanaceae family native of West Indies and introduced to India as an ornamental plant. Because of its fragrant flowers and its moderate size, it is been planted as an ornamental plant 1.
The leaves contain many active constituents which have been used for tropical psoriasis therapy in many parts of the world. Ayurvedic practioners have also started to show interest in the plant. Its leaves are used externally to control itching, psoriasis and patches on skin. Its oil is effective in treating malaria in many African countries. Although leaves are toxic but they contain several active compounds which possess medicinal properties. The plant also shows larvicidal and cardioactive activities 2.
Another plant Cestrum nocturnum L., from the same family is an ornamental plant. Its flower blooms during night time calling it “Night Blooming jasmine”, Night Cestrum and Lady of the Night 3. It is native of tropical and subtropical regions of the world and is found in India too. Its leaves have been used traditionally for treating swellings and burns 4. The oil is effective as its volatile and act as mosquito repellent and have been used in many African countries to cure malaria 5. It is used to treat epilepsy too. Pharmacological studies have shown that it can be used to treat arterial hypertension, and has analgesic, diuretic, antiviral and abortive properties6.
MATERIALS AND METHODS:
Plant Collection and Identification: Fresh plant samples were collected from two different localities. Identification of species was done by comparing with authenticated herbarium specimens, later confirmed with the help of diagnostic keys and morphological descriptions given in various floras. The useful parts such as leaves, stems etc. were separated and preserved for study.
- Anatomical Study of the Part Used: The stems and the leaves of both the plants were fixed in F.A.A. (i.e. Formalin, acetic acid-alcohol 1:1:18) after trimming them to correct dimensions. Hand sections were cut using sharp blade. Thin transverse sections were stained in safranin and then fast green, passed through alcohol grades for dehydration and then mounted in D.P.X. Observations were taken from these sections using light microscope. These sections were also photomicrographed. Special identifying features of the plant part(s) were studied and identified.
- Phytochemical Study: Leaves were washed with a solution of 5% mercuric chloride for 5min and then thoroughly washed with sterile distilled water in order to remove any impurities, shade dried and oven dried and fine powder was made. The solvent extracts were evaporated to dryness in rotary evaporator in distilled water, ethanol and methanol. The dried residues thus obtained were stored in screw capped vials at - 4°C. Phytochemical studies were carried out by following methods:
Test for alkaloids:
- Hager’s test: Test solution was treated with few drops of Hager’s reagent (saturated picric acid solution). Formation of yellow precipitate showed the presence of alkaloids.
- Mayer’s reagent and Wagner’s Reagent: The plant extract was warmed with 2% H2SO4 for two minutes, then it was filtered and few drops of reagents were added separately.
- A) Mayer’s reagent: A creamy white colored precipitate showed the presence of alkaloids.
- B) Wagner’s Reagent: A reddish-brown precipitate appears which also confirmed presence of alkaloids in the extract.
- Test for Tannins: The extract was mixed with basic lead acetate solution and formation of white precipitate indicated the presence of tannins.
- Test for Cardiac glycosides: To the solution of extract in glacial acetic acid solution, few drops of FeCl3 and conc. H2SO4 were observed for the reddish-brown coloration at the junction of two layers and bluish-green color in upper layer.
- Test for saponins: Frothing test: About 5 ml of filtrate was diluted with 20ml of water and shaken vigorously. A stable forth upon standing showed the presence of saponins.
- Test for Steroids and Terpenoids: 4mg of extract was treated with 0.5ml of acetic anhydride and 0.5 ml of chloroform. Then concentrated solution of H2SO4 was added and res violet color was observed for terpenoids and green - bluish color for steroids.
Test for Flavonoids:
- Ferric-chloride test: Few drops of FeCl3 solution were added to the extract which formed the black color indicated the presence of flavonoids.
- Lead-acetate solution test: To the extract was added a few drops of lead acetate (10%) solution which resulted in the yellow precipitate thus indicating the presence of flavonoids.
- C) High Performance Liquid Chromatography: The methanolic leaf extracts were analyzed using HPLC (Shimadzu, 2LC-10 ATVP pumps, SPD-10AVP, UV - visible detector, Rheodyne injector with 50 ?L loop. The data was acquired and processed using Shimadzu LC- solution version 6.42 software. Phenomenex C18 column (250mm×4.6mm, I.D., 5?m) and 4% aqueous acetonitrile containing 0.1% (v/v) phosphoric acid buffered to pH 3.5 with triethylamine was used as mobile phase. The mobile phase was filtered through 0.22?m membrane filter and degassed by sonication for 10 mins. Injection volume was adjusted to 20?L and detection was made at 260nm.
Anatomical Study of the Plants: The comparative account of anatomical features of plant parts such as leaf and stem are shown in the Table 1 and 2.
TABLE 1: COMPARATIVE ANATOMY OF STEM OF TWO SPECIES
|Cestrum diurnum||Cestrum nocturnum|
|Epidermis: It is made up of single layer of
uniseriate elongated rectangular row of cells.
|Epidermis: It is made up of multiseriate
rows of small parenchymatous cells.
|Cortex: It is made up of many layers of
parenchymatous cells along with air cavities.
|Cortex: Epidermis is followed by cortex having
patches of chollenchyma along with parenchymatous cells.
|Endodermis: Cells are compactly arranged in
two three layers around the vascular tissues and
forms circular rings around the vascular tissue.
|Endodermis: It is not very prominent and forms a
wavy ring around the vascular tissues.
|Vascular tissues: Secondary growth is clearly visible,
outer and inner phloem are prominent. Secondary xylem
is visible between phloem region and vessels are large and prominent. Secondary xylem forms an extensive compactly arranged region. Pith is prominent showing round parenchymatous cells. Calcium oxalate crystals are
seen in patches in the pith
|Vascular tissues: Secondary growth is visible with
small outer and inner phloem region. Vessels are small
and pith has compactly arranged small parenchymatous
cells as compared to C. diurnum
TABLE 2: COMPARATIVE ANATOMY OF LEAF OF TWO SPECIES
|Cestrum diurnum||Cestrum nocturnum|
|Epidermis: It is made up of single layer of cells usually
small towards the centre and elongated towards the outside.
|Epidermis: Uniseriate row of
parenchymatous cells are present.
|Collenchyma: Patches of rounded cells below epidermis on
both upper and lower sides.
|Collenchyma: Single layer of collenchymatous
cells is present on both upper and lower epidermis.
|Parenchyma: A layer of parenchymatous
cells surrounding the vascular tissues.
|Parenchyma: Parenchymatous cells are present
around vascular tissues.
|Vascular bundles: An oval-shaped central ring of vascular bundle
is present which has phloem on both the sides and xylem in the
centre, i.e. bi-collateral and open vascular bundle is present.
|Vascular bundles: Stele is crescent-shaped present
in the centre, open vascular bundle is present
|Lamina: Mesophyll is differentiated into spongy and
palisade parenchyma. Palisade parenchyma is visible
towards the abaxial side in 2-3 layers.
|Lamina: Mesophyll is differentiated into
palisade and spongy parenchyma.
Palisade parenchyma is visible
Cestrum diurnum Stem:
FIG. 1: T. S. STEM CESTRUM DIURNUM SHOWING Ep-Epidermis, Co-Cortex, Col- Collenchyma, Pa-Parenchyma, En- Endodermis, Oph- Outer phloem, Sx- Secondary xylem, Iph- Inner phloem, Pi- Pith
FIG. 2: T.S. STEM CESTRUM DIURNUM SHOWING VESSELS AND XYLEM FIBRES
Cestrum diurnum Leaf:
FIG. 3: T.S. CESTRUM DIURNUM LEAF MIDRIB SHOWING Adx- adaxial side, Ep- Epidermis, Co- Collenchyma, Iph- Inner phloem, Xl- xylem fibres, Oph-Outer phloem and Abx- Abaxial side.
FIG. 4: T.S. CESTRUM DIURNUM LEAF LAMINA SHOWING Adx- adaxial side, Ep- epidermis, Pm- palisade mesophyll, Vb- vascular bundles, Sm- Spongy mesophyll and Abx- abaxial side.
Cestrum nocturnum Stem and Leaf:
FIG. 5: T.S. STEM CESTRUM NOCTURNUM SHOWING Ep-Epidermis, Co-Cortex, Col- Collenchyma, Pa-Parenchyma, En- Endodermis, Oph- Outer phloem, Sx- Secondary xylem, Iph- Inner phloem, Pi- Pith
FIG. 6: V.S. CESTRUM NOCTURNUM LEAF MIDRIB SHOWING Adx- Adaxial side, Ue- upper epidermis, Pm- palisade mesophyll, Iph- inner phloem, Xl- xylem, Oph- Outer phloem
Phytochemical Studies: Different phytochemical constituents analysed from two species of Cestrum are given below: The qualitative analysis revealed the presence of alkaloids, glycosides,saponins and flavonoids in all three extracts in both the species.
TABLE 3:PHYTOCHEMICAL STUDIES ON CESTRUM DIURNUM LEAF EXTRACT
+ (Present), - (absent)
TABLE 4:PHYTOCHEMICAL STUDIES ON CESTRUM NOCTURNUM LEAF EXTRACT
Active Principle Analysis By HPLC:
HPLC profiles of both the species: In the present study, it has been observed that in case of Cestrum diurnum maximum peak area was observed as 20530 with retention time 4.345 whereas in case of C.nocturnum the maximum peak area was observed at 808 with retention time 4.340. The peaks were observed by comparing with the reference standard which showed the maximum retention time of 4.578. Hence from the chromatograms it is clearly observed that Cestrum diurnum has maximum peak area with more amount of alkaloid content present as compared to C. nocturnum. One more peak has been identified which is of nornicotine but its has less retention time i.e. 3.796 and 3.604 in C. diurnum and C. nocturnum respectively. The chromatograms of HPLC profile of standard, and both the species have been given in the Fig. 7, 8 and 9 respectively.
TABLE 5: PEAK TABLE
|Sample ID||Area||Concentration||Nicotine present(μg/ml)|
FIG. 7: HPLC CHROMATOGRAM OF NICOTINE(STANDARD)
FIG. 8: HPLC CHROMATOGRAM OF CESTRUM DIURNUM LEAF EXTRACT
FIG. 9: HPLC CHROMATOGRAM OF CESTRUM NOCTURNUM LEAF EXTRACT
DISCUSSION: Qualitative screening of phyto- chemical constituents reveal the presence of alkaloids, flavonoids, saponins, glycosides in all the extracts in the Table 3 and 4 whereas tannins are absent in aqueous and methanolic extracts of Cestrum nocturnum and steroids and terpenoids are absent in aqueous extracts in Cestrum diurnum. Pharmacological studies have demonstrated that the leaf extracts possess nicotine as major alkaloid along with nornicotine which is found to be maximum in Cestrum diurnum as shown in the Fig. 8 and 9 through its peak area when studied through HPLC.These alkaloids possess wide range of medicinal properties such as antibacterial, antiinflammatory, antispasmodic, expectorant and vermifuge properties 8. The anatomical studies of the part(s) used help to identify the distinguishing features. Since the drug is primarily obtained from leaves these have been studied in detail to prevent adulteration of commercial drug by users. Certain diagnostic features of morphology and anatomy have been found useful for the correct identification of species of Cestrum plant in the field to obtain the drug in its purest form.
CONCLUSION: Approaches like screening, phytochemical profiling of these plants helps to find out elite species. The correct identification of the herbal drugs of commerce help to check piracy of these drugs and hence make available true botanicals to consumers and manufacturers of drugs for its correct use as medicine which depends on the exact amount needed to make it more efficient source of medicine for its use in the best possible way.
ACKNOWLEDGEMENT: This research was supported by UGC, New Delhi, India is greatly acknowledged. I am also very thankful to Department of Botany, Panjab University, Chandigarh for providing the necessary facilities.
CONFLICTS OF INTEREST: Nil
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How to cite this article:
Nishtha, Richa and Rao A: Phytochemical analysis and anatomical study of two species of Cestrum from Chandigarh. Int J Pharm Sci Res. 2017; 8(12): 5234-40.doi: 10.13040/IJPSR.0975-8232.8(12).5234-40.
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