1. INTRODUCTION:

Skin aging is a complex biological process that involves many intrinsic factors (genetics, cellular metabolism, hormones, metabolic processes) as well as extrinsic (chronic light exposure, pollution, ionizing radiation, chemicals and toxins) factors.All of these factors result in cumulative structural and physiologic alterations in skin layers and a change in skin appearance, especially on areas exposed to the sun¹. Presently, a variety of aging-related diseases are emerging as the most health threats in most developed countries.

Even though it is not yet possible to modify our genetic background, various anti-aging strategies are presently emerged as healthy lifestyles and therapeutic interventions, aiming to diminish the prevalence of risk factors of poor health². One question is imposing in the age of modern science and technology, with plastic surgery and laser rejuvenation techniques. Is there a place for natural, herbal products?³.

In many cellular processes, Reactive Oxygen Species (ROS) play an important role⁴. As UV radiation is absorbed by the skin, ROS are generated and oxidative stress is triggered. A high level of oxidative damage can lead to many types of damage, including tissue damage, mitochondrial and DNA damage, protein and gene modifications, which can alter protein structure and function⁵. When ROS levels are elevated, these enzymes—Hyaluronidase, Collagenase, and Elastase—are activated, which further contributes to skin aging. Herbal drugs which have an anti-oxidant potential may contribute to inhibit Hyaluronidase, Collagenase, Elastase and Tyrosinase activity which are important for anti aging activity.

India has an ancient (>5000 years old) system of medicine known as Ayurveda, that signifies the science of longevity. It emphasizes dietary and therapeutic approaches for health promotion, disease prevention, and rejuvenation in order to slow the aging process of the body and replenish organ functions. "Rasayana chikitsa" refers to the rejuvenation or restructuring strategies of Ayurveda. The Rasayana ('Rasa': plasma; Ayana: path; which means the path that Rasa takes) group is a group of Ayurvedic medicinal plants used for this purpose.⁶

Amalaki rasayana/AR is derived from the fruits of Phyllanthus emblica or Embilica officinalis; family of Euphorbiaceae, referred to as ‘Amla’ by the local Ayurvedic practitioners as a rejuvenating medicine. Amla fruits are commonly used in Ayurvedic medicine to strengthen the body's defences. There is evidence in Ayurveda literature that Amalaki may have beneficial effects on cancer, diabetes, liver diseases, heart disease, and ulcers, among other ailments. Amla contains emblicanin, gallic acid, ellagic acid, Phyllemblin, quercetin, phyllantine and phyllantidine.⁷ In addition, it contains amino acids, carbohydrates, and vitamin C, showing that it may help cure a wide range of ailments.⁸ As an antioxidant, immunomodulator, antipyretic, analgesic, cytoprotective, antitussive and gastroprotective, AR is used by the Indians. It can also improve memory and lower blood cholesterol levels. There is also evidence that its consumption protects tissues against radiation damage from various scientific studies. In the present study, Amlaki Rasayana (AR) is evaluated for its anti- aging potential by anti- collagenase assay, since this formulation is prescribed traditionally for aging problems besides determining its anti-oxidant activity.

2. MATERIALS AND METHODS:

Extraction:

The marketed Amlaki rasayana (AR) formulation was procured from the Ayurveda shop. The powder sample was macerated with ethanol for about 72h, 48h and 24h. It was then filtered and concentrated in rotary evaporator and stored. This ethanol extract was used for phyto- chemical, invitro anti-oxidant and anti- aging studies.

DPPH free radical scavenging assay:

An ethanol solution (0.2mM) of DPPH was freshly prepared and kept in dark until use. A volume of 1ml of the extract was added into 1ml of DPPH solution. Replacing the extract with solvent solution the control was made. The mixture was incubated for 30min in dark at ambient temperature and thereafter the absorbance at 517nm in UV-Visible spectrophotometer was measured. Quercetin was used as standard for comparison. All determinations were done in duplicate to calculate the percentage radical scavenging activity of the AR extracts.¹⁰

Nitric oxide scavenging method:

This activity was conducted based on Griess assay method. The mixtures of 50,100, 200, 400, 800, and 1000μg (1ml) AR extracts were mixed with 2ml Sodium nitroprusside (10mM in phosphate buffer) and incubated at 37°C for 4 hours. The sample was run above with the blank being replaced with same amount of water. After the incubation period, the above incubated solution was added to 0.5ml Griess reagent. The control solution was prepared without the samples. The absorbance (chromophore formed) was read at 546nm in UV spectrophotometer. Vitamin C was kept as positive control and the results were expressed as percentage inhibition of nitric oxide. All determinations were performed in duplicates.¹¹

Anti- collagenase assay:

About 50 μl of collagenase (1mg/ml) was added to all tubes except blank. TES buffer (50mM) with 0.36mM calcium chloride (pH 7.4) 150μl (blank), 100μl (negative control), 50μl to sample and solvent control tubes. Different concentrations of sample (100 μl) was added to respective tubes.100μl of DMSO (solvent) was added to solvent test tube. After incubating the tubes for 20 min in a water bath at 37°C, 200μl (2mM) FALGPA was added and incubated further for 60 min at 37°C. Adding solution (400μl) containing equal volumes of a 200mM citrate buffer (pH 5) and ninhydrin solution to each tube was done. All tubes were allowed to cool to room temperature following warming in a water bath (100°C) for five minutes, before 50% isopropanol (400μL) was added to each tube. Absorbance was detected at 540 nm. It was then compared with the standard EDTA.¹²

3. RESULTS AND DISCUSSION:

Phytochemical evaluation:

The ethanol extract was evaluated for the presence of Phytoconstituents and the results are given in table 1. The results revealed the presence of alkaloids, flavonoids, tannins, carbohydrates and phenolic compounds.

Table: 1 - Phyto- Chemical Evaluation of ethanol extract of Amalaki Rasayana (AR)

S. No	Constituents	Ethanol extract
1.	Alkaloids	+
2.	Flavonoids	+
3.	Tannins	+
4.	Terpenoids	-
5.	Saponin	-
6.	Carbohydrate	+
7.	Glycoside	-
8.	Phenolic compounds	+

+ Present; - Absent

DPPH radical scavenging activity:

DPPH radical scavenging evaluation of formulation was performed and the results were presented in Table 2.

Table: 2 - Percentage Scavenging Activity of Ethanol Extract of the Formulation by DPPH radical scavenging assay

S. No	Concentration (µg/ml)	Percentage Scavenging
S. No	Concentration (µg/ml)	Formulation	Standard (Quercetin)
1.	50	23.3±0.52	89.09±0.40
2.	100	36.11±0.37	90.475±0.41
3.	250	47.59±0.50	91.28±0.45
4.	500	61.2±0.3	92.55±0.14
5.	1000	80.15±0.04	93.80±0.24

Value represents mean ± standard deviation. IC50 value = 554.2 μg/ml

Nitric oxide scavenging activity:

Percentage Nitric oxide scavenging assay was performed and the results were presented in table 3.

Table: 3 - Percentage Scavenging Activity of Ethanol Extract of the Formulation by Nitric Oxide Scavenging Activity

S. No.	Concentration (µg/ml)	Percentage Scavenging
S. No.	Concentration (µg/ml)	Formulation	Standard (Ascorbic acid)
1.	50	24.4±0.45	79.505±0.36
2.	100	39.1±1.28	82.22±0.38
3.	250	47.0±0.43	85.62±0.52
4.	500	52.6±1.7	87.415±0.37
5.	1000	67.5±1.24	91.515±0.65

Value represents mean ± standard deviation. IC50 value was found to be 432.4 μg/ml

The in- vitro anti- oxidant activity of Amlaki Rasayana (AR) was performed using DPPH and nitric oxide scavenging assay method. The DPPH radical scavenging assay revealed that concentration dependent scavenging activity with the ethanol extract and the results are comparable with standard quercetin. The percentage inhibition was more at 1000 μg/ml of the ethanol extract in Nitric oxide scavenging assay. The IC₅₀values of AR ethanol extract in DPPH and nitric oxide scavenging assay was established to be 554.2 μg/ml and 432.4 μg/ml respectively.

Table: 4 - Anti- Collagenase Activity of traditional formulation

S: No	Concentration (µg/ml)	Percentage Inhibition
1.	50	16.6
2.	100	33.3
3.	250	50
4.	500	83.3
5.	1000	87.4
6	EDTA Positive control	85.9

The anti- aging potential of Amlaki Rasayana (AR) was evaluated by enzymatic method (anti- collagenase assay) and the results are reported in table 4. The results revealed that concentration dependent inhibition was observed with the extract and the IC₅₀ value was reported to be 297.6μg/ml and compared with standard EDTA.

DISCUSSION:

There are two types of skin aging: intrinsic and extrinsic. Extrinsic skin aging or photo-aging is the result of exposure to solar radiation (photo-aging). Intrinsic skin aging or natural aging is mostly driven by changes in skin elasticity over time. Various changes in connective tissue like lipid peroxides, cells contents, enzymes, and reactive oxygen species (ROS) are formed when the skin is exposed to ultraviolet light and these contributes physical changes to the skin. Lipid peroxides can be metabolised to produce secondary products that damage the extracellular matrix (ECM), while ROS are thought to be involved in conditions such as arthritis, diabetes, cancer and loss of skin elasticity.^13-15

As a branch of Ayurveda-based practice, Rasayana manages the rejuvenations and regenerations, immunostimulations as well as the processes of healthy aging. A variety of organic products, including fruits, herbs, spices, and flavors are blended to form rasayanas of different kinds and are conventionally used to improve health.¹⁶In the Charaka Samhita, rasayana is believed to boost mental capacity, eyesight, energy, and brightness.The Indian customary system of medicine utilizes AR as a cardiovascular, cerebral, and digestive tonic, typically made from the fruits of Amalaki (amla; Indian gooseberry, Phyllanthus emblica).^17-22

In the present study we evaluated the Amlaki Rasayana (AR) for the phyto chemical and anti- aging studies. The alcoholic extract was prepared with rasayana and used for phyto- chemical, anti- oxidant and anti- aging studies. The phyto- chemical screening revealed the presence of tannins, alkaloids and flavonoids. The in- vitro anti- oxidant activity of Amlaki Rasayana (AR) was carried out by DPPH and Nitric oxide radical scavenging assay method. The DPPH assay revealed the concentration dependent scavenging activity with the extract and the results are comparable with standard quercetin. The percentage inhibition was more at 1000 μg/ml of the ethanol extract in Nitric oxide scavenging assay. The in- vitro anti- aging potential was evaluated by anti- collagenase assay method using FALGPA as substrate. The ethanol extract exhibited significant inhibition on collagenase enzyme which was comparable to standard EDTA. The ethanol extract of Amlaki Rasayana(AR) showed significant anti- oxidant and anti- aging activity which may be attributed to phyto-constituents such as flavonoids, alkaloids and tannins. This study offers scientific validation of Amlaki Rasayana(AR) for anti- aging activity and can be recommended to be taken internally for aging problems.

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Received on 02.03.2021 Modified on 10.05.2022

Research J. Pharm. and Tech 2023; 16(8):3521-3524.

DOI: 10.52711/0974-360X.2023.00581