INTRODUCTION:

India has been recognized as treasure or rich house of medicinal plant species since several years. One of the medicinal plants i.e. Rhododendron is a naturally occurring plant in a higher altitudinal region which possesses some well-being benefits, like prevention and treatment of diseases associated with detoxification, dysentery, bronchitis, asthma and constipation. Rhododendron (family Ericaceae) is a Greek derived word “Rodo” means rose and “dendron” means trees, first studied in 1837 by Carl Linnaeus. Rhododendron originally found in the valleys of Himalayas Kashmir Manipur Assam in India and in Bhutan as well. The most widely distributed species in the western to eastern Himalayan region of India is Rhododendron arboreum¹.

It is known to be the world's largest Rhododendron and holds Guinness World record. They have bright red flowers, and appear as small evergreen trees with somewhat crooked trunks. They have soft bark generally used as fuel and timber. This species is considered special because it beholds various medicinal benefits and economic values. In Uttrakhand these are widely popular for its processed juice and squash from flowers. They are so beautiful and attention seeking to the visitors due to which this flower has been and titled the regional flower of Himachal Pradesh India and the national flower of Nepal. Rhododendron naturally possess many health benefits such, as they are helpful in treatment and prevention of so many diseases associated with dysentery, heart, diarrhea, detoxification, fever, inflammation, bronchitis, constipation and asthma. their have a high antioxidant activity². The new or the leaves young leaves are used in elevation of headache. the wood or the bark of this plant is used for making khukri handles, gift boxes, gun stocks, posts, packsaddles and etc. the flowers are limitedly available but these flowers are used in food and pharmaceutical sectors with few limitations. They also contain many secondary metabolites like flavonoids, saponins, alkaloids, glycosides, tannins, steroids, and phlobatannins which are essential for plant survival and also play many significant roles in human health^3,4. There are various phytochemicals that have been isolated, identified and reported till now from various different parts of Rhododendron arboreum^4,5.

One of the species of Rhododendron i. e. Rhododendron rawatii having shiny green leaves with glabrous young shoots. R. rawatii appears from shrubs to small trees not more than 4.5m tall. Their flowering period ranges from March- May and capsule dehiscence occurs from October onwards. it has thin and peeling bark, which appears from reddish to whitish in color. their leaves are clustered at the bottom of the branches. Their leaf size appears from 11-13cm which are shiny green having cottony hairs at the surface^4-6. The flower appears whitish to pink and red in color, they are loosely arranged on the cluster of leafs, their glabrous pedicle is 6-13mm long. the flowers has dark pink to brown patches all over the surface. This species is specifically found in Kedarnath wild life sanctuary along the timber line zone. One of the other population is found Chhirkila in Chhipla-Kedar in about 3120m from sea level Pithoragarh district of Uttrakhand. They grow in the area where the daily mean air temperature is always below 0⁰C in maximum time in a year, and the soil remains acidic in nature (4.3 - 5.0) and remains under the snow for nearly 3-4 months of a year. The objective of our study is to compare and evaluate the immunopharmacological activity of Rhododendron arboreum and Rhododendron rawatii.

MATERIALS AND METHODS:

Sample collection:

The sample (leaves and flowers) of Rhododendron arboretum (3100-3350m from sea level) and Rhododendron rawatii (3200-4300m from the sea level) has been collected from Tungnath in Rudrapryag district of Uttarakhand. The collected leaves and flowers were washed thoroughly in running fresh water and then left for drying in shade or air dry for around 2-3 weeks. The leaves and flowers when dried are crushed in a blender and kept in air tight jar or small bag in refrigerator at 4⁰C as a storage for future use. Rhododendron arboreum and Rhododendron rawatii (leaves and flower) were identified andauthenticated by a Dr. AB Bajpai, Department of Botany, DBS PG College, Dehradun and was collected in April 2022.

Preparation of leaves and flower extract:

Leaves and flowers (Rhododendron arboreum and Rhododendron rawatii, 10g) were dried in a shady area and separately cut into small pieces, and prepared powdered form. For these studies, methanol used as solvent system for extraction of antioxidant and antimicrobial derived compounds from Rhododendron species. Leaves and flowers were separately extracted with solvent i.e. methanol (100%; 1 L) for 48-72h. After incubation, extract of leaves and flower were then concentrated at 68°C under reduced pressure pertaining to obtain the flower and leaves residues in the form of crude extract. Finally, both these extracts (10 mg/ml) were kept at 4°C after filtration of crude extract using whatmann filter paper for analyzing its phytoconstitutents using standard procedures and also determined its antioxidant, antimicrobial and anti-inflammatory based studies.

Anti-oxidant activity using DPPH Assay:

One of the standard method i.e. DPPH radical scavenging assay for measuring the antioxidant activity in case of leaves and flowers extract of Rhododendron arboreum and Rhododendron rawatii. In this experiment, methanolic DPPH solution (24mg/100ml; 3ml) was added along with leaves or flower extract using different concentrations (1-10mg/ml; 100μl). Methanol used as control for these studies. Incubate these samples for 30-45 min and absorbance was measured at 517nm. Ascorbic acid with different concentrations (1-10mg/ml) was used as positive control^7,8. For measuring its radical scavenging activity of DPPH and readings were calculated on the basis of this equation:

DPPH Scavenging effect (%) = [A0- A1)/ A0] ×100

Where, A0 means control absorbance and A1 means absorbance of methanolic leaves or flower extract or Ascorbic acid.

Estimation of total flavonoid content:

To estimate the concentration of total flavonoid content in leaves and flowers of Rhododendron arboreum and Rhododendron rawatii. In this study, plant extract (0.3 ml) is added with methanol (30 %; 3.4ml) and NaNO₂ (0.5 M; 0.15ml) and its mixture was allowed to react for 5-6min and then AlCl₃solution (0.3 M; 0.15ml) is added. The absorbance was then measured after 15 min. At the wavelength of 506 nm, different concentrations of ascorbic acid were used as a standard to quantify the total flavonoids. The measurement was carried out in triplicates. The total flavonoid content was expressed in milligram^9,10.

Estimation of total phenolic content:

Leaves and flowers of Rhododendron arboreum and Rhododendron rawatii were being estimated for total phenolic content by using Folin- Ciacolteu method. A total of 1ml extract solution and 1ml of Folin-Ciocalteu reagent is mixed and kept for 5minutes, following which 7% of Na₂CO₃(10ml) were added to the mixture and then add deionized distilled water (13ml). The mixture was homogenized and was incubated for 30min in the dark area. The absorbance was then measured at 750nm and measured its total phenolic content and these readings were calculated based on the calibration curve (using ascorbic acid). This measurement was carried out in triplicates^9,10.

Antimicrobial activity:

In this assay, we determined its antimicrobial activity of Rhododendron species (Rhododendron arboretum and Rhododendron rawatii) especially leaves and flowers against gram positive and gram negative bacteria was carried out by utilizing agar well diffusion method. For these studies, 100microliter of bacterial cell suspension was spread equally over the nutrient agar plate. Laterthe wells were punched into the agar plate using a sterilized cork borer. A 50 microliter ofsample extract of different concentrations were added to each well. Agar plates were thenincubated at 37ºC for 24hours. The tests were performed and prepared in triplicates. The results were then noteddown keeping standard, control, methanol and comparing with methanolic leaves and flower extract. Results were expressed in the form of millimeters (mm)^11.

Statistical analysis:

The results were expressed in the form of Mean ± S.E. and compared the data using statistical software (one-way ANOVA).

RESULTS:

Phytochemical analysis:

For estimation of secondary metabolites in leaves and flowers extract of Rhododendron rawatii and Rhododendron arboreumas shown in Table 1. The results of these studies showed that leaves of Rhododendron rawatiis howed higher amount of flavonoid, tannins and saponin as compared to Rhododendron arboreum. Saponins are absent in the flower extract of both species of Rhododendron.

Anti-oxidant activity:

Rhododendron rawatii methanolic extract scavenged DPPH radical in such a way that it showsthe higher activity as compared to control and Rhododendron arboreum. The leaf extracthas a very good scavenging activity in case of Rhododendron rawatiibut less as compared to standard (Ascorbic acid) as shown in the Fig. 1. This confers the Rhododendron rawatii extract hasa rich composition of Phenolic and flavonoid providing them the higher potential of scavengingfree radicals at higher doses (10mg/ml;100µl). The antioxidant activity is normally based on the free radical scavenging activity.The DPPH assay is based on the ability to reduce the violet color which shows anti-oxidantactivity. Higher change in the color indicated highest antioxidant activity i.e. Rhododendron rawatii extractis expected to have a good amount of flavonoid and phenolic compound.

Fig.1. Antioxidant activities of leaves and flower extract from Rhododendron species (rawatii ad arboreum) various concentrations using DPPH radical scavenging activity.

Each value represents in the form ofMean ± SE (n = 3). One-way ANOVA is applied for this test. *P<0.05; **P<0.01 and ***P<0.001

Flavonoid and phenolic content:

For determining the content of flavonoid and phenolic content in case of Rhododendron arboretum and Rhododendron rawatiias shown in Fig. 2. The results of these studies showed that leaves extract of Rhododendron rawatiiat higher concentration having higher flavonoid and phenolic content as compared to flowers extract of Rhododendron arboreum.

Table 1: Phytochemical analysis

S. No.	Bioactive compounds	*Rhododendron arboreum,* Leaves	*Rhododendron rawatii* Leaves	*Rhododendron arboreum,* flower	*Rhododendron rawatii,* flower
1.	Terpenoids	+ + +	+ + +	+	+
2.	flavonoids	+ + +	+ + + +	+	+
3.	Tannins	+ +	+ + +	-	+
4.	Saponins	+ +	+ + +	-	-
5.	Alkaloids	+	+ +	+	+

Fig.2. Flavonoid and phenolic content in leaves and flower extract of Rhododendron rawatii and Rhododendron arboreum.

Each value represents in the form ofMean ± SE (n = 3). One-way ANOVA is applied for this test. *P<0.05; **P<0.01 and ***P<0.001

Table 2: Antimicrobial activity of leaves and flowers extract of Rhododendron rawatii and Rhododendron arboreum against test organisms

S. No.	Test organisms	Zone of inhibition (mm)
		Rhododendron rawatii		Rhododendron arboreum		Penicillin	Methanol
		Leaves	Flowers	Leaves	Flowers	Penicillin	Methanol
1	Staphylococcus aureus	18 ± 1.66**	13 ± 0.94	16 ± 1.78*	11 ± 1.08	21 ± 2.24***	3 ± 0.24
2	Pseudomonas aeruginosa	15 ± 0.88*	14 ± 0.66*	16 ± 1.56*	10 ± 0.98	17 ± 1.82**	3 ± 0.16
3	E. coli	16 ± 1.22*	12 ± 1.34	13 ± 1.44	8 ± 0.78	14 ± 2.82*	2 ± 0.08

Values were expressed as Mean ± S.E.*P<0.05; **P<0.01 and ***P<0.001

Anti-microbial activity:

Table 2 shows that the Leaf extract of Rhododendron rawatii has a higher anti-microbial activity for gram positive and gram negative bacteria than compared to the flower extracts. The activity was directly proportional tothe concentration used in the test. Among the two samples of Rhododendron arboretum and Rhododendron rawatii, as Rhododendron rawatii has a higheranti-microbial effect over Gram positive and gram negative bacteria.

DISCUSSION:

Uttarakhand, an Indian state located in the Himalayan region with a great natural diversity, occupies 17.3 percent of India's total land area, with 92.57 percent of the territory covered by hills and 7.43 percent by plains. Authors reported ethno-medicinal applications of 102 plant species from 48 families from the four districts of the state, namely Champawat, Almora, Pithoragarh and Bageshwar. Adhikari et al., researched the condition and distribution pattern of therapeutic plants at the Wildlife Institute of Dehradun in Uttarakhand, recording 605 plants from 94 families. Several studies were conducted related to medicinal plants and its major objective is to replace the allopathic medication because of high price value. In this regard, many people especially in villages relied on herbal medicines having less side effects. Indigenous peoples still maintain and orally transmit a large quantity of traditional knowledge regarding the usage of medicinal plant species^12,13. The main objective of our studies is to evaluate the compared the species of Rhododendron arboreum and Rhododendron rawatii (leaves and flowers) using methanol as solvent system and evaluate its antioxidant, anti-inflammatory and antimicrobial activity.

Natural antioxidants were reported from vegetables, fruits, spices, leaves, cereals etc. and played an important role especially in human healthcare sector. The most striking ability of an antioxidant from medicinal plants pertaining to trap free radicals which are responsible for causing infectious diseases. In literature, antioxidant substances from medicinal plants such as flavonoids, polyphenols, phenolic acids and scavenge free radicals (i.e. peroxide, hydro peroxide, and lipid peroxyl)have been reported and showed several immunpharmacological activities. The most familiar examples of antioxidants i.e. flavonoids and phenolic compounds have been recorded and may be considered as effective agent for various infectious diseases. In this regard, we determined the flavonoid and phenolic compound from these Rhododendron species and evaluated its antioxidant property using DPPH. This assay (DPPH) is used to measure the antioxidant activity of Rhododendron species which have ability to transfer electrons. It’s a chromophore which is stable radical cation possessing purple color^14,15. So antioxidant compounds, having capacity to donate an electron to DPPH radical cation, cause a discoloration of the solution when added in the solution having DPPH radical in it. These studies showed that Rhododendron rawatii having higher antioxidant activity in case of leaves extract as compared with Rhododendron arboreum (leaves and flowers) and flowers of Rhododendron rawatii.

Another major objective of our study was to examine theleaves and fruit extracts of Rhododendron arboretum and Rhododendron rawatiis elected based on traditional medicinal use for measuring its antimicrobial activity. The antimicrobial activity of Rhododendron species has been analyzed using disc diffusion assays and showed its activity against gram positive and gram negative bacteria.With referenceto bacterial growth inhibition, it is not unexpected to see unsatisfactory activity of flowers extract in case of Rhododendron arboretum and Rhododendron rawatii. However, disc diffusion results can be informative as a basic starting point to identifying species with potential antibacterial activity.

CONCLUSION:

The following study was performed to compare Rhododendron arboreum with Rhododendron rawatii with all aspects. The following study declares that the Rhododendron rawatii showed significant differences in the capacity of antioxidant in Rhododendron extracts were measured with DPPH method, which also showed that the capacity of Rhododendron rawatii is higher than Rhododendron arboreum. The total phenolic content gave a very strong correlation to antioxidant capacity and antimicrobial activity against bacterial strains.

REFERENCES:

1. Joshi RK. Joshi BC. Sati MK. Chemical and Chemotaxonomic Aspects of Some Aromatic and Medicinal Plants Species from Uttarakhand: A Review. Asian J Pharm Tech. 2014; 4(3): 157-162. doi.org:10.5958/j.0975-4261.4.2.002.

2. Parween SA. Farooqui M. Asema SUK. Farooqui M. Evaluation of Analytical Parameters of Some Medicinal Plants. Asian J Research Chem. 2010; 3(4): 850-852.doi.org: 10.5958/0974-4150.

3. Srivastava P. Rhododendron arboreum: an overview. Journal of Applied Pharmaceutical Sciences. 2012; 2:158-162.

4. Madhvi SK. Sharma M. Iqbal J. Younis M. Phytochemical analysis, Total flavonoid, Phenolic contents and antioxidant activity of extracts from the leaves of Rhododendron arboreum. Research Journal of Pharmacy and Technology. 2020; 13(4): 361-369. doi.org: 10.52711/0974-360X.2022.00816.

5. Popescu R. Loop B. The genus Rhododendron: an ethanopharmacological and toxicological review. Journal of Ethnopharmacology. 2013; 1(10): 42-62. doi.org:10.1016/j.jep.2013.02.022.

6. Painuli S. Rai N. Kumar N. Gas chromatography and mass spectrometry analysis of methanolic extract of leaves of Rhododendron arboretum. Asian Journal of Pharmaceutical and Clinical Research. 2016; 9(1): 74-82.

7. Kashyap P. Anand S. Thakur A. Evaluation of antioxidant and antimicrobial activity of Rhododendron arboretum flowers extract. International Journal of Food Fermenting Technology. 2017; 7(1): 123-128.doi.org: 10.5958/2277-9396.2017.00013.7

8. Bhandari L. Rajbhandari M. Isolation of quercetin from flower petals, estimation of total phenolic, total flavonoid and antioxidant activity of the different parts of Rhododendron arboretum smith. Journal of Scientific World. 2014; 12(12): 245-78. doi.org/10.3126/sw.v12i12.13569.

9. Rafi M. Febriany S. Wulandari P. Hsuparto I. Rahayu S. Total phenolics, flavonoids, and anthocyanin contents of six variety Rhododendron from Indonesia and Evaluation of their antioxidant activities. Journal of Applied Pharmaceutical Science. 2018; 8(9): 49-54. doi.org:10.7324/JAPS.2018.8908.

10. Madhvi S. Sharma M. Iqbal J. Younis M. Phytochemistry, traditional uses and pharmacology of Rhododendron arboretum: A review. Research Journal of Pharmacy and Technology. 2019; 12(9): 3618-360.doi.org:10.5958/0974-360X.2019.00785.6.

11. George J. Bioactive screening and antimicrobial activity of selected three medicinal plants on chosen microbes. Research Journal of Pharmacy and Technology. 2014; 7(11): 1264-1269. doi.org: 10.5958/0974-360X.

12. Duraisamy A. Narayanaswamy N. Balakrishnan KP. Antioxidant and Anti-Tyrosinase Activity of Some Medicinal Plants. Research J Pharmacognosy and Phytochemistry. 2011; 3(2): 86-90.doi.org: 10.5958/0975-4385.

13. Maharaja P. Sengottuvel T. Aarthi A. Gopalasatheeskumar K. Review on Antioxidant and Hepatoprotective activity of Medicinal plants against Paracetamol Induced animal model. Res J Pharmacognosy and Phytochem. 2020; 12(2): 114-119. doi.org: 10.5958/0975-4385.2020.00020.5.

14. Konduri MKR. Uppuluri KB. Chintha R. Mulla S, Peruri R. In-vitro Antimicrobial Activity of Four Indigenous Medicinal Plants Belonging to Bapatla, A.P. Research Journal of Pharmacy and Technology. 2010; 3(2): 461-465. doi.org: 10.5958/0974-360X.

15. Kulkarni A. Kale M. Sarode S. Firke S. Firke B. Warke P. Antimicrobial Activity of Some Important Medicinal Plants of India against Some Plant and Human Pathogens. Research Journal of Pharmacy and Technology. 2010; 3 (3): 924-926. doi.org: 10.5958/0974-360X.

16. Momin MAM. Mamunur R. Kaniz FU. Sohel R. Phytochemical Screening and Investigation of Antioxidant and Cytotoxicity Potential of different extracts of selected Medicinal Plants of Bangladesh. Research Journal of Pharmacy and Technology. 2013; 6(9): 1042-1050. doi.org: 10.5958/0974-360X.

Received on 27.07.2022 Modified on 15.12.2022

Research J. Pharm. and Tech 2023; 16(12):5765-5769.

DOI: 10.52711/0974-360X.2023.00933

¹Department of Microbiology and Department of Biotechnology,

Graphic Era (Deemed to be) University, Dehradun.

1Department of Microbiology and Department of Biotechnology,

Graphic Era (Deemed to be) University, Dehradun.

¹Department of Microbiology and Department of Biotechnology,