FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

1. TITLE OF THE INVENTION:
AN HERBAL ANTI AGING FORMULATION
2. APPLICANT:

(a) NAME : AVANCÉ PHYTOTHERAPIES PVT. LTD.
(b) NATIONALITY : AN INDIAN COMPANY
(c) ADDRESS : 204, CIRCLE-P BUILDING
PRAHLADNAGAR
S. G. HIGHWAY
AHMEDABAD-380054
GUJARAT, INDIA

3. PREAMBLE TO THE DESCRIPTION
PROVISIONAL

The following specification describes the invention. þ COMPLETE

The following specification particularly describes the invention and the manner in which it is to be performed.

The present invention relates to an herbal anti aging formulation comprising of alcoholic powder extracts from genus Terminalia, Glycyrrhiza and Areca.

BACKGROUND OF INVENTION
A skin, the largest organ in human body, is considered to be pivotal because it is involved in a variety of physiological functions such as protection of several organs from environmental stimulations, offering a barrier for prevention of water and useful constituents in body from release, regulation of body temperature, respiration and excretion.
The aging process is taking place continuously in metabolism, and even gets rapid process in state of disease or stress. Skin is exposed to ultraviolet rays that make continuous chemical reaction. In addition, smoking, pollution, and virus infection became major cause of skin aging. The physiological alterations in skin, with aging, include: (a) decrease of the thickness of the epidermis, dermis and sub-dermal tissues; (b) decrease of barrier function due to alteration of lipid composition and content in lipid barrier resulting in skin drying; and (c) elicitation of freckles, pigmentation and various skin lesions.
The researches in the cosmetic art have been made to prevent and treat the skin aging-associated phenomena such as wrinkles, decreased elasticity, freckles and drying. In order to retard skin aging, the cosmetic compositions conventionally contain moistening agent, anti-inflammatory agent or nutritive additives for tissues with degraded cross-linkages of elastin and collagen. Anti aging formulations mainly contain mercury (II) chloride or ammoniated mercury, hydroquinone, arbutin, alpha hydroxy acid (AHAs), tretinoin and kojic acid. Mercury has been banned in most countries (Europe 1976) (USA 1990) for use in anti aging because it accumulates on skin and it can have the opposite results in the long term. Hydroquinone has been banned in some countries (e.g. France) because of fear of a cancer risk. While a controversial research has suggested that kojic acid may have carcinogenic properties in large doses. However, such compositions generally exhibit a limitation in the fundamental retardation of skin aging.
US6682763 discloses a skin beautifying, anti aging formulation containing olive extract, whitening agents, antioxidants, anti-inflammatory agents, cell activators, UV-screening agents, blood circulation-promoters and humectants. The invention claims to be used as a skin beautifying agent, anti aging, skin whitening and external agent for skin. The use of various ingredients in the formulation makes it complex.
WO2009130344 discloses a personalized cosmetic formulation formed by retinoic acid, anti-inflammatory agent, depigmenting agent, anti-oxidant and any vitamin. The invention includes using indomethacin, hydroquinone, kojic acid, mequinol, phytic acid, alpha-arbutin, lipolic acid, lycopene, resveratrol, coenzyme Q10, pycnogenol, N-acetyl glucosamine, L-carnosine, taurine, ascorbic acid, etc. The chemicals used in the formulation are the ones mainly used in any skin cosmetics available in market and certain are found to be carcinogenic in nature.
WO2009138801 discloses a cosmetic formulation for beautifying skin and uses retinoic acid as main ingredient with various other ingredients palmitoyl tripeptide, caprooyl tripeptide, ethyl bisimino methyl guaiacol manganese chloride, algisium C.
The anti aging formulations incorporating above described ingredients exhibit poor stability, so that it’s long action cannot be expected and thus its application range to cosmetics is very limited. Also the ingredients used in these products like Kojic acid, hydroquinone and mercuric compounds, are recently found to be carcinogenic on long term use. Therefore, there remains a need in the art for an inhibitor capable of fundamentally preventing the degradation of elastin and collagen. Hence, we need the system that can restrain to delay and inhibit the skin aging not just inside the body but also on the skin.
The conventional anti aging creams available in market contain chemical compounds that harm the skin and can lead to permanent damage. The anti aging formulations available in prior art do not give anti aging, anti wrinkles, skin hydration, skin moisturising and freckles reduction in one herbal compound. The present invention is effective against skin drying, fine wrinkles, thin and fragile skin, transparent skin and loss of firmness, dull and lifeless appearance and sagging skin.

OBJECT OF THE INVENTION
The main object of the present invention is to provide an herbal anti aging formulation containing alcoholic powder extracts from genus Terminalia, Areca and Glycyrrhiza.
Further the object of the present invention is to provide formulation which is selected from the group of solution, suspension, emulsion, paste, ointment, gel, cream, lotion, oil, powder, spray, tablet, capsule and sterile injectable solution.
Other object of the present invention is to provide an herbal anti aging formulation with no side effects or negative effects on skin.
Another object of the invention is to provide an herbal anti aging formulation for reducing fine wrinkles, freckles, skin drying, for providing moisture to skin, preventing sagging of skin, loss of firmness, dull and lifeless appearance.
Further another object of the invention is to provide an herbal anti aging formulation which enhances collagen, elastin and hyaluronic acid levels in the skin and reduces MMP-1 (matrix metalloproteinase-1) levels thus gives the desired effect.
Yet another object of the invention is to provide an herbal anti aging formulation which is effective and economical.

SUMMARY OF INVENTION
The present invention deals with an herbal anti aging formulation containing alcoholic extracts from powder of genus Terminalia, Glycyrrhiza and Areca in quantities of 35- 45% w/w, 35- 45% w/w and 45- 55% w/w, respectively. The extract is prepared from the stems, roots, rhizomes, leaves, fruits, flowers, pericarp or seeds of the plants. The formulation can be in form of solution, suspension, emulsion, paste, ointment, gel, cream, lotion, oil, powder, spray, tablet, capsule and sterile injectable solution. The herbal formulation has anti aging and skin moisturizing properties and reduces wrinkles and freckles on skin. The formulation has no negative effects on skin and is completely safe and effective on use.

DETAILED DESCRIPTION OF THE DRAWINGS
Fig. 1 shows effect of present formulation on viability of Human Foreskin Fibroblast (HFF-1) cells after 48 hr treatment.
Fig. 2 shows the effect of present formulation on proliferation of HFF-1 cells.
Fig. 3 shows the protective effect of present formulation on oxidative stress generated by H2O2.
Fig. 4 shows effect of present formulation on cell cycle.

DETAILED DESCRIPTION
The nature of invention and the manner in which it is performed is clearly described in the specification. The invention has various components and they are clearly described in the following pages of the complete specification.
Anti aging is a controversial topic as it is closely intertwined with the detrimental effects on health, identity, self image, racial supremacy and colonial mentality.
The conventional anti aging creams available in market contain chemical compounds that harm the skin and can lead to permanent damage. Also the chemical compounds like kojic acid, hydroquinone and mercuric compounds used in conventional formulations are found to be carcinogenic in nature which is dangerous on long term use.
The present invention enhances collagen, elastin and hyaluronic acid levels in the body and reduces MMP-1 level in the body.
The present invention is effective against skin drying, fine wrinkles, thin and fragile skin, transparent skin and loss of firmness, dull and lifeless appearance and greater visibility of skin pores and sagging of skin.
The present invention comprises of mainly powder extracts form Myrobalan, Liquorice, Areca nut and excipients.
Myrobalan is dried ripe matured fruit belonging to the family Combretaceae and having main chemical constituents chebulic acid, chebulagic, chebulinic, ellagic and gallic acids. It is commonly known as Baheda or Hardea. Myrobalan used in the present invention can be selected from the group of Belliric myrobalan (Terminalia bellirica), Chebulic myrobalan (Terminalia chebula) and Arjun myrobalan (Terminalia arjuna). It is mainly found in Gujarat. It is mainly used as an astringent, laxative, stomachic, tonic, anthelmintic, used in treatment of piles and external ulcers and commercially used in dyeing and tanning industry.
Liquorice is the dried root or stolon belonging to the family Leguminosae, having main chemical constituents glycyrrhizin, glycyrrhizinic acid and carbenoxolone. It is commonly known as Jethimadha or Mulethi. Liquorice in the present invention is selected from Mediterranean Liquorice (Glycyrrhiza glabra), North American liquorice (Glycyrrhiza lepidota), Russian liquorice (Glycyrrhiza echinata) and Chinese liquorice (Glycyrrhiza uralensis). It is found in Gujarat. It is mainly used as an expectorant and demulcent, as a flavouring agent in formulations for nauseous drugs, beverages, confectionary and pharmaceuticals, for treatment of peptic ulcer and rheumatoid arthritis, inflammation and Addison’s disease, used as an antispasmodic, an anti-inflammatory agent and also used as foam stabilizer.
Areca nut is dried ripe seed of Areca catechu belonging to the family Palmae, having main chemical constituents arecoline, arecaidine, guvacine and guvacoline. It is commonly known as Supari or Betel nut. It is found all over India. It is mainly used as parasympathomimetic, an anthelmintic, a vermicide and taenifuge in veterinary practice.
In the present invention alcoholic powder extracts from genus Terminalia, Glycyrrhiza and Areca are prepared by employing methods already known in the prior art.
In the present invention the extract is prepared from the stems, roots, rhizomes, fruits, leaves, flowers, pericarp and seeds of the plants from the genus Terminalia, Glycyrrhiza and Areca.
Various formulations are prepared from alcoholic powder extracts from genus Terminalia, Glycyrrhiza and Areca which are made into formulation selected from solution, suspension, emulsion, paste, ointment, gel, cream, lotion, oil, powder, spray, tablet, capsule and sterile injectable solution by conventional methods.
In-vitro tests like determination of non cytotoxic concentration (Fig. 1), effect of present formulation on fibroblast proliferation (Fig. 2), protective effect of present formulation on viability against oxidative stress (Fig. 3), and effect of present formulation on cell cycle (Fig. 4) were conducted and compared to conventional chemical moieties having anti aging properties.
The test results reported that, as directed in Fig. 1, pre-treatment of HFF-1 cells with different concentration of present formulation (0.1 -100 µg/ml) for 48 hr resulted in more than 90% viability as compared to control cells, hence the present formulation was found to be non cytotoxic.
As directed in Fig. 2, HFF-1 cells were treated with non cytotoxic concentrations of present formulation (5- 25 µg/ml) for 72 hr and the effect on fibroblast proliferation was assessed. Treatment of HFF-1 cells with present formulation resulted in stimulation of cellular proliferation upto 39.1 % at 5 µg/ml and 49.7 % at 10 µg/ml respectively as compared to control. Vitamin C (100 µM), which was used as a positive control, demonstrated an increase in cell proliferation by 20.4 %.
As directed in Fig. 3, HFF-1 cells were pre-treated with non cytotoxic concentrations of present formulation and (5 - 25 µg/ml) for 24 hr and their effect on retention of viability against H2O2 induced oxidative stress was determined. Only 23% of HFF-1 cells were left viable upon treatment with 2mM H2O2. However, prior treatment of HFF-1 cells with present formulation resulted in 2.03 fold increase in cell viability (from 23 % to 46.82 %) at 5 µg/ml as compared to H2O2 treated cells. Vitamin C (100 µM), which was used as a positive control demonstrated an increase in cell viability from 23% (H2O2 treated) to 74.1 %.
As directed in Fig. 4, treatment of HFF-1 cells with present formulation led to higher percentage of proliferating cells in S-phase as compared to control cells. Percentage of cells in S-phase was increased from 4.43 % (control cells) [Fig. 4 (a)] to 7.94 % and 6.03 % at 25 µg/ml [Fig. 4 (c)] and 50 µg/ml [Fig. 4 (d)] respectively. As directed in Fig. 4 (b), vitamin C (100 µM), which was used as a positive control demonstrated an increase in proliferating cells in S-phase from 4.43 % (control cells) to 8.05 %.
There was anti aging, anti wrinkles, skin hydrating and comparative reduction of freckles and increase in extra cellular matrix proteins (ECM).
The formulation contains 35- 45% w/w standardized alcoholic powder extract from genus Terminalia, 45- 55% w/w standardized alcoholic powder extract from genus Areca, 35- 45% w/w standardized alcoholic powder extract from genus Glycyrrhiza and excipients quantity sufficient.

The invention is illustrated more in detail in the following examples. The examples describe and demonstrate embodiments within the scope of the present invention. These examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as many variations thereof are possible without departing from the spirit and scope.

Example 1:
Sr. No. Ingredients Quantity (mg)
1 Terminalia chebula 95
2 Areca catechu 145
3 Glycyrrhiza glabra 95
4 Excipients q.s.

Example 2:
Sr. No. Ingredients Quantity (mg)
1 Terminalia chebula 100
2 Areca catechu 150
3 Glycyrrhiza glabra 100
4 Excipients q.s.

Example 3:
Sr. No. Ingredients Quantity (mg)
1 Terminalia chebula 105
2 Areca catechu 155
3 Glycyrrhiza glabra 105
4 Excipients q.s.

The tests for the present formulation were conducted on the Human Foreskin Fibroblast (HFF-1) cell line in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 15% FBS (Fetal Bovine Serum) under growth conditions of 37°C, 95% humidity and 5% CO2. The tests conducted were estimation of extra cellular elastin levels, estimation of hyaluronic acid levels, estimation of collagen levels and estimation of MMP-1 levels. The results obtained are as given below in as per the tables.

Table 1: Effect of present formulation and its ingredients on elastin level in HFF-1 cells after 24 hr of treatment, compared to water control
Groups Concentrations (µg/ml) Elastin Levels
Example 1 Example 2 Example 3
Present Formulation 25 289.2 ? 394.2 ? 350.1 ?
50 603.8 ? 583.0 ? 553.9 ?
Terminalia chebula extract 7 109.3 ? 105.1 ? 129.7 ?
14 110.6 ? 117.3 ? 132.2 ?
Areca catechu extract 10 283.9 ? 270.3 ? 300.6 ?
20 176.1 ? 197.9 ? 217.8 ?
Glycyrrhiza glabra extract 7 200.4 ? 185.4 ? 190.3 ?
14 206.7 ? 218.6 ? 203.5 ?

Table 2: Effect of present formulation and its ingredients on hyaluronic acid level in HFF-1 cells after 24hr of treatment, compared to water control
Groups Concentrations (µg/ml) Hyaluronic acid level
Example 1 Example 2 Example 3
Present Formulation 25 185.9 ? 173.9 ? 200.6 ?
50 134.6 ? 131.8 ? 142.1 ?
Terminalia chebula extract 7 123.8 ? 153.7 ? 145.9 ?
14 130.1 ? 128.6 ? 114.3 ?
Areca catechu extract 10 163.7 ? 145.1 ? 163.8 ?
20 125.3 ? 130.8 ? 125.5 ?
Glycyrrhiza glabra extract 7 164.4 ? 170.2 ? 154.7 ?
14 125.2 ? 127.1 ? 115.8 ?

Table 3: Effect of present formulation and its ingredients on collagen level in HFF-1 cells after 24hr of treatment, compared to water control
Groups Concentrations (µg/ml) Collagen level
Example 1 Example 2 Example 3
Present Formulation 25 134.3 ? 165.1 ? 152.5 ?
50 123.6 ? 142.2 ? 139.4 ?
Terminalia chebula extract 7 122.3 ? 158.2 ? 142.1 ?
14 118.9 ? 138.3 ? 122.3 ?
Areca catechu extract 10 122.4 ? 124.4 ? 112.2 ?
20 121.7 ? 136.8 ? 117.9 ?
Glycyrrhiza glabra extract 7 120.1 ? 115.2 ? 110.7 ?
14 121.8 ? 119.1 ? 123.6 ?

Table 4: Effect of present formulation and its ingredients on MMP-1 in HFF-1 cells after 24hr of treatment, compared to water control
Groups Concentrations (µg/ml) MMP-1 level
Example 1 Example 2 Example 3
Present Formulation 25 57.2 ? 50.6 ? 60.3 ?
50 55.4 ? 49.5 ? 52.7 ?
Terminalia chebula extract 7 19.9 ? 18.5 ? 17.4 ?
14 32.6 ? 28.3 ? 35.9 ?
Areca catechu extract 10 8.8? 7.3 ? 19.5 ?
20 21.1 ? 15.4 ? 20.1?
Glycyrrhiza glabra extract 7 18.3 ? 21.2 ? 16.8 ?
14 30.5 ? 27.1 ? 30.2 ?

In the test above, the anti-aging potential of the herbal formulation and its individual ingredient was evaluated on ECM markers in HFF-1 cell lines. There was a remarkable increase in the level of extracellular proteins which are important in preventing age related symptoms like wrinkles, sagging, loss of water and fragile skin. There was a significant increase in ECM molecules like insoluble proteins, collagen and elastin as well as insoluble proteins like hyaluronic acid. Areca catechu extract increased elastin level (270.3%) but was significantly less when compared to the formulation (583.0%). Similarly, Terminalia chebula extract and Glycyrrhiza glabra extract increased collagen (158.2%) and hyaluronic acid level (170.2%) respectively but their individual effect was less when compared to effect of the present herbal formulation on collagen (165.1%) and hyaluronic acid (173.9%) levels. On treatment with the present herbal formulation there was 50% reduction in levels of matrix metalloprotenases-1 (enzymes which are involved in breaking down ECM proteins), this reduction was much significant when compared to that of Terminalia chebula (28.3%), Areca catechu (15.4%) and Glycyrrhiza glabra extract (27.1%).
Based on the above study it can be concluded that the present herbal formulation has significant anti-ageing effect and this potential is collective and synergetic effect of its individual component. Also that the present invention is effective against skin drying, fine wrinkles, thin, fragile and transparent skin, loss of firmness, dull and lifeless appearance and sagging skin.
Although the preferred embodiment as well as the preparation and use have been specifically described, it should be understood that variations in the preferred embodiment could be achieved by a person skilled in the art without departing from the spirit of the invention. The invention has been described with reference to specific embodiments which are merely illustrative and not intended to limit the scope of the invention as defined in the claims.

We claim,
1. An herbal anti aging formulation containing powder extracts of plants belonging to genus Terminalia, Glycyrrhiza, Areca and excipients.
2. The herbal anti aging formulation as claimed in claim 1, wherein the plant belonging to Terminalia genus is selected from the group of Terminalia chebula, Terminalia bellirica and Terminalia arjuna.
3. The herbal anti aging formulation as claimed in claim 1, wherein the said plant belonging to Glycyrrhiza genus is selected from Glycyrrhiza glabra, Glycyrrhiza lepidota, Glycyrrhiza echinata and Glycyrrhiza uralensis.
4. The herbal anti aging formulation as claimed in claim 1, wherein the said plant belonging to Areca genus is Areca catechu.
5. The herbal anti aging formulation as claimed in claim 1, wherein the stems, roots, rhizomes, flowers, leaves, fruits, pericarp, seeds of plants is used to prepare extract.
6. The herbal anti aging formulation as claimed in claim 1, wherein alcoholic powder extract of the plants is used.
7. The herbal anti aging formulation as claimed in claim 1, wherein the powder extract of Terminalia is 35- 45% w/w.
8. The herbal anti aging formulation as claimed in claim 1, wherein the powder extract of Glycyrrhiza is 35- 45% w/w.
9. The herbal anti aging formulation as claimed in claim 1, wherein the powder extract of Areca is 45- 55% w/w.
10. The herbal anti aging formulation as claimed in claim 1, wherein said formulation is in form of one selected from the group consisting of solution, suspension, emulsion, paste, ointment, gel, cream, lotion, powder, soap, spray, tablet, capsule and sterile injectable solution.
11. The herbal anti aging formulation as claimed in claim 1, wherein the formulation is effective against skin drying, fine wrinkles, thin and fragile skin, loss of firmness, dull, lifeless appearance and sagging skin.
12. The herbal anti aging formulation, which is substantially as herein described with reference to the foregoing description and the accompanying examples.