INTRODUCTION:

Niacinamide (also known as nicotinamide, 3-pyridinecarboxamide) is the physiologically active form of niacin or vitamin B3. Niacinamide, a derivative of niacin, has the ability to treat some skin conditions including aging skin. It has anti-inflammatory properties, which makes it effective for treating acne. It may also be beneficial for treating other inflammatory skin conditions such as psoriasis and rosacea. Acne is a skin condition characterized by excess sebum production and irregular shedding of dead skin cells. The dead skin cells and sebum clog hair follicles causing inflammation, which niacinamide helps to reduce. In the body, it is converted to the co-factors NADH and NADPH that are involved in many biochemical reactions. NAD+ and NADPH levels in skin cells decline with age.

Thus, supplementing skin with the precursor to these vital co-factors has the potential to provide appearance benefits to aging skin. Since niacinamide penetrates the skin’s surface readily, it is bioavailable from topical application for targeted delivery to specific skin sites. However, since niacinamide is an important precursor of NADH and NADPH, it has been postulated that topical application of niacinamide can promote this reported broad spectrum of activity through local correction of homeostatic balance of these two nucleotide coenzymes.

Physiological Role of Niacinamide:

Research by Oblong et show that Nicotinamide Coenzymes in Skin are depleted with age; Niacinamide can help normalize this imbalance. Aged Fibroblasts Secrete Less Collagen than Young Cells. Niacinamide can Stimulate New Collagen Synthesis by Up-regulates Epidermal Ceramide Synthesis. and also regulate Sebaceous Lipid and Acne. Shen et al demonstrated the ability of niacinamide to protect cultured normal human keratinocytes against reactive oxygen species induced by UVC irradiation or exposure to hydrogen peroxide. This supports published data showing the ability of both topical and oral niacinamide to prevent UV-induced immunosuppression.

EXPERIMENTAL:

Instrument used for Analysis:

1. Camag HPTLC system with camag TLC scanner 4 with,

i. TLC aluminium sheet Silica gel 60 F 254 plate (10/10cm).

ii. CamagLinomet 5 applicator (syringe size 100ul)

2. Ultra violate spectroscopy. (Shimadzu model 1800)

Software used:

LC Solution (Shimadzu high performance liquid chromatography).

Preparation of herbal distillate:

Take required qty of DM water. Heat the water at about 100°C. Take formula qty of herbs. Ensure that heating is stopped before the immersion of herbs. Soak the herbs into the DM water. Add MP and PP. Closed the vessel with a loosely fitted lid and leave as such for 10 to 12 hrs. Filter it. Put the filtrate into the distillation unit for distillation. Collect the distillation.

Preparation of water phase:

Transfer hot DM water into a batch praparation kettle through filter and add Propylene glycol, PEG 32, MP (as per formula quantity) start homogenizer and side scraper for mixing at 80 -85°c.

Preparation of oil phase:

Take formula quantity of Oil Phase material in the oil melting kettle heat it at about 80 -85°C

Mixing of oil phase into water phase for emulsification:

Transfer gradually into the mixing kettle through 100 mesh filter and continue homogenizing. Start high speed mixing and scraping arrangement. The rest quantity of hot DM water transfer to the oil phase melting kettle; ringe well and transfer to the batch mixing vessel (To flash the oil transfer line) Mix well. Dissolve the formula qty of KOH in DM water (Previously heated and then cooled) Immediately add this solution in a slow stram into the batch mixture and mix water phase, KOH and oil phase material for 30 mins. Add the material Flocare ET-58 in the same order and mixed well. Immediately add the premix solution of Tapioca starch (Autoclaved for 1.5 hrs at 15 lb Press.), previously heated and Cooled DM water and continue high speed mixing and homogenizing. Stop homogenizer after 15-20 mins and start water circulation for cooling. Add the mixture of DC 9050 and DC 3031, at 63° - 60 ° C and continue mixing with side scrapping and start cooling. Dissolved the formula qty of niacinamide in DM water (at 65-70°C). At 52-50°C add the mixture into the mixing vessel and continue mixing and cooling. Dissolve the Neolone 950 in DM water (Previously heated) add the mixture into the mixing vessel at 50-48°C and continue mixing and cooling. Immediately add the mixture of Rose Water. Immediately add the herbal distillate Continue mixing.

Determination of marker ingredient Niacinamide by HPTLC:

Preparation of HPTLC standard curve:

0.02 g of Niacinamide std was weight accurately in a 10 ml volumetric flask, dissolved it by methanol: water (1:3) and make up to the mark with same solvent mixture. Filter the solution by 0.45u. 1ul,2ul, 4, ul, 6ul above solution was injected (contain 6ug, 12ug, 24ug, 36ug) on TLC aluminium sheet Silica gel 60 F 254 plate (10/10cm) by using Camag Linomet 5 applicator (syringe size 100µl).

Table 11: Serial dilution to produce different concentrations of niacinamide

Volume of stock taken in µl	1	2	4	6
Amount of niacinamide in ug	6	12	24	36

After drying the TLC plate put into the mobile phase and run time is 10min. Retention factor, peak height, peak area was measured by Scanning the plate using camag TLC scanner 4 at 254nm.

Estimation of niacinamide in sample:

Separation of oil phase:

5g o/w emulsion containing niacinamide (1.5 % w/w) was taken in a separating funnel add 100 ml of water,20ml of petroleum benzene was mixed to it shake vigorously and released pressure frequently. Stand the mixture for 5 min, after complete separation of organic phase take the water phase, repeat the same procedure for 3 times. After removing the oil phase through organic solvent filter the water phase and stored.

Niacenamide estimation From Water phase of the cream:

The above solution (concentration 50mg/ml) was taken in a volumetric flask. 3µl, 5µl, 6µl sample was apply by CamagLinomet 5 applicator (syringe size 100µl).

Table 2: Serial dilution to produce different concentrations of sample

Volume of stock taken in µl	3	5	6
Amount of niacinamide in ug	150	250	300

After drying the TLC plate put into the mobile phase and run time is 10min. Qualitative and quantitative estimation is done by observing Retention factor, pickhight, pick area measured by Scaning the plate using camag TLC scanner 4 at 254nm.

RESULT AND DISCUSSION:

Table 3: Formulation design for o/w cream(500)

Ingredients	Amount(gm)
Glycyrrhizaglabra	0.95
Alovera powder ext	0.005
Nicinamide	7.5
Methyl paraben	1.03
Propyl paraben	0.01
Propylene glycol	2.5
PEG—32	2.5
DC-200/350	2.5
Potassium hydroxide	3.5
Ingredients	Amount(gm)
Propylene glycol	5
Tapioca Starch	15
DC 9040	2
Silicon DC- 3031	5
Nicinamide	7.5
DM water	300
Neolone 950	0.5
Stearic Acid	50

Quantitative estimation of Niacinamide (By HPTLC):

Niacinamide in cream is esmimated by high performance thin layer chromatography.

Identification of Niacinamide in sample.

The TLC Plate shows (Figure-1) the standard and sample point for Niacinamide, With same Rf value. It help to identify the presence of niacinamide in sample.

Figure 1: TLC plate of NIacinamide

Quantitative estimation of Niacinamide in Sample

Standard curve of niacinamide:

λmax is of Niacinamide was determined from U.V. Visible spectrophotometer by scanning through the entire range (190-1100). The λmax was found to be 254 nm against the reagent blank. To determine the amount of active ingredient present in the formulation at different concentration standard curve were calibrated. Here standard curve of niacinamide was estimated in HPTLC.In this method area and height of the HPTLC spectrum curve was measured in different concentration.

Table 5: Estimation of % of Niacinamide in sample (According to area)

Track	Batch	volume applied (µl)	Amount applied (ug)	Area	Rf	Amount of Niacinamide (ug)	% of Niacinamide	Std dv	Avg %
1	1	3	150	10185.98	0.51	2.581	1.71
2	1	5	250	11987.69	0.51	3.037	1.21	0.3536	1.46
3	2	3	150	9073.22	0.51	2.299	1.53
4	2	5	250	10385.28	0.51	2.631	1.25	0.1980	1.40
5	3	3	150	8571.20	0.51	2.171	1.44
6	3	5	250	9831.62	0.52	2.591	1.33	0.0778	1.38

Table 6: Estimation of % of Niacinamide in sample (According to Height)

Track	Batch	volume applied(µl)	Amount applied (ug)	Height	Rf	Amount of Niacinamide (ug)	% of Niacinamide	Std dv	Avg
1	1	3	150	304.94	0.51	2.730	1.82
2	1	5	250	351.22	0.51	3.146	1.25	0.4031	1.53
3	2	3	150	261.26	0.51	2.340	1.56
4	2	5	250	289.18	0.51	2.590	1.03	0.3748	1.30
5	3	3	150	216.77	0.51	1.942	1.10
6	3	5	250	238.62	0.52	2.137	1.00	0.0707	1.14

According to area % of niacinamide in batch 1,2 and 3 are respectively 1.46, 1.40 and1.38.

According to height % of niacinamide in 3 different sample from batch no 1, 2 and 3 are 1.53, 1.30 and1.41.

According to formulation 1.5% of Niacinamide is given in this o/w base cream. We analyzed the % of Niacinamide in 3 batch of the cream. And the calculated amount of Niacinamide from HPTLC analysis are shown in table.

Table 7: % of Niacinamide In different Batch.

Batch	% of Niacinamide (according to Area)	% of Niacinamide from (according to Height)	Average	Std dv
1	1.46	1.53	1.49	0.1159
2	1.40	1.30	1.35
3	1.38	1.14	1.26

The % of Niacinamide in 3 batch are 1.49,1.35 and 1.26, and standard deviation is 0.1159.

CONCLUSION:

Niacinamide based topical dermatological product has been prepare and evaluated successfully. Active marker % of Niacinamide in 3 batch is in between 1.2 to 1.5. All the data is in between the specific limit that is 1.2-1.63. Free fatty acid and total fatty matter of cream from all layer are also in acceptable limit. So the product quality and uniformity is satisfied and the process also well established.

ACKNOWLEDGEMENT:

The Authors are thankful to the management authoritarians of Emami Limited Kolkata for providing necessary facilities to carry out this study.

CONFLICT OF INTEREST:

This artical has not published before and it is not under consideration for publication in any other journal.

REFERENCES:

1. Shinoda, K., and Saito, Hj. Colloid and Interface Sci. (1968)

2. Stjepanović-Veselčić, L. Hypericaceae. In Flora of Serbia, 1st ed.; Josifović, M., Ed.; SANU: Belgrade, Serbia, 1972; Volume 3: pp. 104-125.

3. Griffith, R.E. Medical Botany, 1st ed.; Lea and Blanchard: Philadelphia, PA, USA, 1847; pp. 156-158.

4. Sarić, M. Medicinal Plants of SR Serbia, 1st ed.; SASA: Belgrade, Serbia, 1989; pp. 315-317.

5. British Herbal Medicine Association. British Herbal Pharmacopoeia; British Herbal Medicine Association: London, UK, 1996.

6. Matts PJ. A review of the range of effects of niacinamide in human skin. IFSCC Mag. 2002; 5(4): 285-289.

7. Mawatari, K., Iinuma, F., Watanabe, M., 1991. Determination of Nicotinic Acid and Nicotinamide in Human Serum by High-Performance Liquid Chromatography with Postcolumn Ultraviolet=Irradiation and Fluorescence Detection, Analytical Science, 7,733-736.

8. A Review of the Range of Effects of Niacinamide in Human Skin Paul J. Matts1), John E. Oblong 2) and Donald L. Bissett2)

9. Statutory Order of the Danish Ministry of Environment and Energy, No. 303 of 18th May 1998, on Cosmetic Products.

Received on 24.02.2019 Accepted on 12.04.2019

Asian J. Pharm. Ana. 2019; 9(2):62-66.

DOI: 10.5958/2231-5675.2019.00013.9

(According to Area)				(According to Height)
Track	Rf	Amount(ug)	Area	Track	Rf	Amount(ug)	Height
1	0.52	4.00	15785.92	1	0.52	4.00	446.45
2	0.52	6.00	19680.35	2	0.52	6.00	530.43
3	0.52	8.00	22882.25	3	0.52	8.00	587.41