A Study of the Hair Shampoo and Conditioner Formulations towards the Effect of Preventing Dust Deposition with the Use of Moringa Oleifera Seed Extract Xiaowei Chang

Henkel(China) Investment Co., Ltd. Building No.8, 398 Songhu Road, Shanghai, 200433, P.R.China Abstract Air pollution, for example the airborne dust particles, is a severe public concern in China by posing threat to the human health and also attracting people’s attention for the impact on skin/hair care. Hair shampoo products are well recognized for removing the grease or particulate pollutants accumulated from the external environment or body secretion. We conducted a series of tests on how the hair treated with normal shampoo prevents and removes the dust deposition. The study result suggested the normal shampoo composed of sodium laureth sulfate and disodium cocoamphodiacetate as major surfactants has limited effect to protect the bleached hair fiber against the dust.. The known coagulant, Moringa Oleifera seed extract, was incorporated into the same shampoo base to improve the dust resistance of human hair. We were able to demonstrate efficacy on bleached Asian hair, by the means of gloss, combing forces, scanning electron microscope, and panel evaluation. The effect was also proven in a hair conditioner system.

Introduction Nowadays, the shampoo usage is widely accepted as routine procedure for consumer to clean their hair. The composition generally includes at least one ionic surfactant, e.g. SLES, for hair cleansing to remove grease or dust resulted from external environment, or self-secreted sebum/dandruff for example. It may contain one or more amphoteric surfactants to boost the foam while buffering the cleansing to achieve a low- moderate caring level. With the evolving of social environment, the developing countries of high population, in recent decades, are facing the air pollution issue which has been encountered in the developed countries, such as blowing dust and dust storm. Those micro sized dust particles are also the carrier of polycyclicaromatic hydrocarbens, which is well known as the organic pollutant with various toxicity1 PM 10/2.5 (particles with a diameter of 10/2.5 m or less) is one important index used for monitoring the air quality as they are the predominant particulates. According to previous studies, most of the dust sample mass are composed of clays, silicon-rich and calcium-rich particles, such as quartz, feldspar and mica as high as >90%.23 Thus the common shampoo as of today - the most popular hair cleansing format is questioned, as an upcoming consumer need, to be able to resist or attract the dust deposition on hair. The dust resistance is more desired in some emerging market where people has less hair washing frequency, while the airborne particulates are of major concerns. Nevertheless, due to its strong emulsifying/cleansing property, shampoo tends to be the most challenged hair application format for active delivery. In our study illustrated below, the Moringa oleifera(MO) seed extract was employed in the shampoo vehicle to evaluate the anti-dust effect of the final composition. The Moringa oleifera(MO) plant seed have multiple benefits well known in medicinal field, as well as industrial applications. For example, MO seed contains a natural organic coagulant which proves more cost effective than commonly used aluminum and iron salt while more environmental friendly. 45 Water treated with MO seed extract produces less sludge volume compared to alum. An additional benefit of using coagulants derived from MO is that a number of useful products may be extracted from the seed. In particular, edible and other useful oils may be extracted before the coagulant is fractionated. Residual solids may be used for animal feed and fertilizer. The MO seed extract we used here contains cationic protein as main composite (0.5 w.t.%) with molecular weight distribution as 80% of average 13,000 daltons, 20% of average 6,000 daltons. Those proteins are believed as the coagulant actives for water treatment. The extract is an aqueous extraction solution and it is easy to incorporate into any water based system.

Materials and methods. 1. Materials: 1.1 Bleached Asian hair, obtained by bleaching the Asian virgin black hair (15cm length, 2g) using the protocol below: a) Prepare the bleaching solution as 17 % active matter of hydrogen peroxide, adjust pH to 9.4 with ammonium hydroxide solution. b) Pour bleaching solution 200 g onto 20 hair tresses. c) Use a flat dyeing brush to distribute carefully the bleaching solution over the strands and make sure that no tangles are induced in the strands, complete application within 1 minute. d) Cover the tray and incubate for 30 minutes at room temperature. e) Rinse hair tress with shower head tap water (38 °C, maximum flow speed) to remove most of the oxidizing solution for 3 minutes. Then repeat the bleaching procedure one more time with fresh bleaching solution. (After bleaching twice, rinse with shower head tap water and comb with the coarse side for 3 min.) f) Soak hair in 1000ml tap water, strands have to be covered with water at r.t., for 4x15minutes; change water after 15 minutes and rinse hair with shower head tap water for 1 minute.

1.2 Hair shampoo and conditioner preparation. The normal shampoo, active shampoo 2% or 4% were prepared based upon the base composition in Table 1. The process starts from dissolving the ionic surfactant in the water, followed by other ingredients from high to low concentration. MO seed extract active was added in the end of process. The normal conditioner, active conditioner 1% were prepared based upon the base composition in Table 2. The process includes the emulsifying at high temperature, followed by adding other minor ingredients at lower temperature as required. MO seed extract active was added in the end of process at room temperature. All above mentioned samples were adjusted with the pH range from 4~6.

Table 1. The shampoo base used to prepare normal shampoo and active shampoos.

INCI % Sodium 2-Laureth Sulfate 10.80 PEG-55 Propylene Glycol Oleate 0.10~0.30 Laureth-2 0.30~0.60 Glycerin 0.10 Lactic acid 0.02~0.05 Na-benzoate 0.50 Disodium Cocoamphodiacetate 3.00 PEG-7 Glyceryl Cocoate 0.50 Polyquaternium-10 0.50~0.80 PEG-7 Glyceryl Cocoate 0.50~0.80 NaCl 0.50~1.00 D．I. water To 100 As MO seed extract required

Table 2. The conditioner base used to prepare normal conditioner and active conditioner 1%.

INCI %

Cetearyl alcohol 5.80

Polyquaternium-87 3.00~4.00

Isopropyl myristate 1.50 Distearoylethyl Hydroxyethylmonium 0.50~0.80 Methosulfate Stearamidopropyl dimethylamine 0.50~0.80

Phenoxyethanol 0.40

Methylparaben 0.30

Polyquaternium-37 0.20~0.50

D-panthenol 0.10~0.20

Dioctyl carbonate 0.20~0.50

D.I. water To 100

MO seed extract As required

1.3 ISO 12103-1, A1 ultra-fine dust was used for dust application. This dust was composed of different elements, with particle sizes from 1~20 m as showed in Table 3 and Table 4. Table 3. the elemental composition of ISO 12103-1, A1 dust.

Composition % Composition %

Si02 68 - 76 Ca0 2.0 - 5.0

Al203 10 - 15 Mg0 1.0 - 2.0

Fe203 2 - 5 Ti02 0.5 - 1.0

Na20 2 - 4 K20 2.0 - 5.0

Table 4. the particle size distribution of ISO 12103-1, A1 dust.

m Quantity Distribution% 1 1.0 – 3.0 2 9.0 – 13.0 3 21.0 – 27.0 4 36.0 – 44.0 5 56.0 – 64.0 7 83.0 – 88.0 10 97.0 – 100 20 100

2. Residue combing work measurement. This method is to measure the combing forces and then the length of strands curves are integrated to obtain the combing work. The equipment used is Zwick/Roell Z2.5 material testing machine. Detailed procedure includes: The bleached Asian hair(15cm/2g) was measured of the combing work before any treatment. The hair tresses were then rinsed under tap water followed by treatment with 1g of shampoo for 4 minutes in total, including application and incubation steps. The treated hair tresses were at last rinsed under tap water for 1 minute. This shampoo application and rinsing process were repeated once more before leaving the wet hair dry out in open air. The combing work of the hair tresses after shampoo application was measured and the residue combing work was calculated as % of after-shampoo treatment versus no treatment. The hair treated afterwards exposed to dust with different levels as shown in Table 5. The combing work of the hair tresses after dust exposure was measured and the residue combing work was calculated as % of after-dust versus no treatment.

3. Hair gloss measurement. The hair tresses were mounted on a sample holder in a shape of cylinder with 10 cm diameter, ensuring the hair is bound with flat and smooth surface. Define the specular reflection and diffuse reflection by polarization system of Visia CR skin analysis imaging system. The hair luster along hair tress were analyzed by scanning across highlighted and dark areas of the resultant image using image analysis software. The luster parameter were calculated using two different empirical models.6

Nc and LReich-Robbins two parameters were thus obtained to represent the gloss characteristics of bleached hair tresses after dust exposure. Empirical Equation of Luster Analysis: S  D Nc  Model of Nickerson: S S L  ReichRobbins D *W Model of Reich and Robbins: stamm 1/ 2 S-the area under the specula curve D-the area under the diffuse curve Dstamm-the area under a straight line obtained by connecting the first and last point of the specular reflection curve W1/2-the width of the specula peak at half of its maximum intensity

4. Scanning electron microscopic measurement. Zeiss Supra 55VP Field Emission Scanning Electron Microscope was used to capture the SEM pictures. Leica EM SCD500 High vacuum sputter coater was used to coat Pt film with the thickness of around 5nm on single hair fiber.

5. Panel evaluation of hair gloss and combility. Hair tresses treated by shampoo/conditioner using abovementioned method in 2 (for both virgin and bleached Asian hair), were dried out and used for pair comparison. Combility and hair gloss were voted by over 13 panelists, and the results were justified with P value ≤0.05 at least.

6. Dust quantification by analyzing SiO2 amount on hair by using Inductively Coupled Plasma Optical Emission Spectrometer (ICP). Equipment: ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer) PerkinElmer Optima 2100DV; Microwave digester with temperature indicator and controller: MDS-6; Teflon tube/Quartz cell with cap-25mL capacity. Detail procedure of sample preparation and testing are as following: Preparation of calibration standards then run the analysis of those standard samples using 2% nitric acid as blank and plot a calibration curve. Digest the hair with dust in the Teflon vessel using microwave digester. Quantitatively transfer the solution to volumetric flasks (polypropylene) by rinsing the tube with several aliquots of distilled water and dilute up to the volume with distilled water. Filter the solution through filter paper. Discard initial few mL of filtrate and collect the remaining filtrate in another flask. Analyze the solutions using ICP-OES following the below mentioned instrumental parameters: Neb0.8L/min Aux0.2L/min Plasma15L/min Wavelength: Si 288.16nm Inject the reagent blank solution and subtract the peak intensity from the sample The level of SiO2 in the sample can be calculated using the formula: Si 25 60 SiO2% =  W 10000 28

7. Dust application chamber and dust densities level for dust deposition. Dust box used is ESON ESR698 sand/dust test machine. And the dust density employed for different testing purposes are shown as in Table 5, with the exposure temperature at around 25°C. Hair tresses were randomly placed inside the dust test machine hold on a stand 25 cm high.

Table 5. The dust density levels for different test purposes. Dust level Dust density Exposure time Test purpose 1 150g/m3 2 min Combing force measurement 2 200g/m3 3 min Gloss measurement and ICP 3 500g/m3 5 min SEM and Panelist evaluation

Discussion The tests of shampoo containing MO seed extract are focused on the repelling efficacy towards the dust particles close to the reality, so ISO 12103-1, A1 dust we chose here is regarded as closest to the current PM10/2.5 particulates in terms of composition as well as particle size distribution. The negative impact of the small particulates for the human hair is well recognized. For example, dull appearance due to their properties of light absorption or reflection. Hair smoothness will be also dramatically affected, such as driving up the overall surface friction, and the combing force. Therefore, the gloss and combility as two key parameters in evaluating hair caring properties were selected to assess the effect of shampoo on dust resistance. SEM is an approach to qualitatively visualize the dust deposition on single hair fiber. To quantify the amount of dust deposited on hair treated with conditioner, SiO2 w.t.% (versus the weight of hair tress) is selected in ICP analysis as the key index since it is the most abundant substance of ISO 12103- 1, A1 ultra-fine dust.

1. Comb work of hair tresses treated by shampoos and followed by dust exposure. One of the important influences of dust deposition on hair is the resulted combing work increase. We started the combing test with the shampooed hair tresses before dust exposure(Figure 1). The bleached hair with the shampoo base treatment showed the combing work reduction to 45.3%. However, the addition of MO seed extract affords a negative impact on combility with 71.0% residue combing work, which is higher than that of shampoo base treated hair tresses. The hair tresses with both shampoo application together the non-treated tresses were exposed to the dust application, at a level of 150g/m3 for 2 minutes. We found that lower level dust of exposure is not helpful to tell the difference between two shampoos, while higher level of dust exposure will lead to the combing work increase dramatically, which is hard to be detected by the combing test machine. As shown in Figure 1, the shampoo base treated hair tresses after exposed to the A1 dust 150g/m3 for 2 minutes, residue combing work increased to the same as non-treated ones, while the active shampoo (2% MO seed extract) treatment reduced the residue combing work to the degree lower than non-treated or normal shampoo treated hair tresses. This implies the less amount of A1 dust deposited on the tresses treated with active shampoo (2% MO seed extract). It is also worthy to note that more dust may be deposited on normal shampoo treated tresses than the ones treated by nothing, since the residue combing work of the former one after dust exposure increase as the same as the latter one.

Figure 1. Residue combing work% of the hair tresses before or after dust exposure versus before any treatment.

2. Gloss measurement and quantification result by Model of Nickerson and Model of Reich and Robbins. This is known that the surface deposition of micro-sized particulates e.g. ZnO will dull the hair and reduce the gloss proved by the calculated luster parameters.7 The hair glosses of non-treated hair tresses, normal shampoo treated and active shampoo treated ones were therefore measured by capturing the specular and diffuse light images (Figure 2.) at the specular and diffuse modes. Dust was applied to the hair tresses at 200g/m3 for 3 minutes. A plot of luminance as a function of distance along each hair tress is then provided for both the specular and diffuse light images. Then use the image analysis software to calculate the two luster parameters Nc and LReich-Robbins (Table 6.).

a) b) c) d) e) f) Figure 2. The specular light image of the hair tress before/after(left/right) dust exposure: a) treated by nothing; b) treated by normal shampoo; c) treated by active shampoo 2%; The diffuse light image of the hair tress before/after(left/right) dust exposure: d) treated by nothing; e) treated by normal shampoo; f) treated by active shampoo 2%.

The calculation of Nc is based upon both the specular and diffuse light images while LReich-Robbins is only achieved from the analysis of specular image and is highly relevant to the width of the luminance peak plotted. The dust deposition can increase the light band width with gloss peak shifting due to the reflection of dust particles, thus leads to an obvious reduction in LReich-Robbins. Before the dust application, we compare the luster parameters Nc mainly, and found the non-treated hair tresses are a little glossier than other two. After dust exposure, all three tresses showed reduction in both the luster parameters Nc and

LReich-Robbins. It was noted that LReich-Robbins of the normal shampoo dropped more significantly than other two with a dropped peak intensity. It suggests the highest dust deposition on the hair tress treated by normal shampoo. This result is consistent with that from combing force measurement. It was hypothesized that the residue from shampoo may induce the dust absorption on the hair fiber – even with 2% MO seed extract this effect can hardly be totally diminished.

Table 6. The luster parameters Nc and LReich-Robbins for the hair tresses treated by different methods, before and after dust exposure.

No treatment Normal shampoo Active shampoo 2% Parameters Before After Before After Before After

Nc 0.6348 0.598255 0.616047 0.584051 0.625622 0.587427

LReich-Robbins 0.102354 0.055826 0.097362 0.035989 0.114129 0.050499

3. Scanning electron microscopic studies of hair tresses treated by different methods. SEM study can reflect the dust deposition straight forward, which is used herein to qualify the active’s effect toward anti-dust. To be able to distinguish the difference between active/normal shampoos, 4% of MO seed extract was added into the shampoo base instead of 2%. The anti-dust effect was well demonstrated by Figure 3d, where the hair fiber shows less dust particles absorbed onto the hair fiber treated by active shampoo 4%. It is noteworthy to point out the coverage of the hair fiber by dust is more severe for normal shampoo treatment than no-treatment.

a) b)

c) d)

Figure 3. Scanning electron photomicrographs of bleached Asian hair: a) without any treatment; b) exposed to dust without any pre-treatment; c) exposed to dust after normal shampoo washing for 2 times; d) exposed to dust after active shampoo 4% washing for 2 times.

4. Gloss:and combility evaluation by panel. Apart from equipment measurement of gloss and combility, panelists were asked to evaluate the same attributes in the lab test. To enable an apparent differences between three treatments, which can be easily perceived, a highest level of dust exposure is employed at 500g/m3 for 5 minutes. The higher the score is rated, more positively the panelists vote for the attribute. The result in Table 7 is quite obvious with significant difference of P<0.01, showing that normal shampoo treatment did not improve the gloss and combility after maximum dust exposure. This is in accordance with our hypothesis that normal shampoo may contribute some residue which attracts the dust adhered to the hair.

Table 7. Gloss and combility of bleached hair tresses without treatment or treated by normal shampoo evaluated by panel No treatment Normal shampoo P Gloss 1.0 0.0 <0.01 Combility 1.0 0.0 <0.01

We also included active shampoo 4% in following test, to see the effect of higher dosage of MO seed extract. It is interesting to note that active shampoo with 2% MO seed extract performs best, on the Asian bleached hair (Table 8.). Active shampoo 4% is still better than normal shampoo, but lose compared to active shampoo 2% on both gloss and combility. Based upon the worse performance of active shampoo 4% together with the combility equipment measurement result before-dust, MO seed extract is deduced to make the hair coarser at higher dosage.

Table 8. Gloss and combility of bleached hair tresses treated by normal shampoo and active shampoo 2% evaluated by panel Normal Active Pa Active Pb shampoo shampoo 2% shampoo 4% Gloss 0.1 2.0 <0.01 0.9 <0.01 Combility 0.2 1.9 <0.01 0.9 <0.01

a. Between groups of active shampoo 2% and normal shampoo. b. Between groups of active shampoo 4% and normal shampoo.

5. The delivery of MO seed extract from shampoo to virgin hair. In the meantime, we evaluated the gloss and combility of the virgin hair tresses treated by different shampoos, including the non-treated ones, by the panel test (Table 9. and Table 10.). The virgin hair tresses were exposed to the same level of dust(500g/m3 for 5 minutes) as the bleached one. Their SEM microphotos were also achieved for a visual comparison. It is observed that normal shampoo may help resist the dust deposition thus increase the gloss/combility, if compared with the non-treated virgin hairs. Contrary to the positive effect on bleached Asian hair, MO seed extract in the shampoo did not show this benefit to virgin hair. There is no significant difference observed except the combility of active shampoo 4% treated hair Vs. norm shampoo treated one. This active induces a coarser surface on virgin hair, similar with what it does on bleached hair. Moreover, the dust amount difference reside on the hair fibers was not visibly distinct from the three SEM micrographs of hair tresses treated with dust exposure in Figure 4.

Table 9. gloss and combility evaluated by panel for non-treated and normal shampoo treated hair tresses. No treatment Normal shampoo P Gloss 0.2 0.8 0.05 Combility 0.2 0.8 0.05

Table 10. gloss and combility evaluated by panel for normal shampoo and active shampoo 2% treated hair tresses. Normal Active Pa Active Pb shampoo shampoo 2% shampoo 4% Gloss 1.3 0.8 0.11 0.9 0.43 Combility 1.7 1.2 0.14 0.1 <0.01

c. Between groups of active shampoo 2% and normal shampoo. d. Between groups of active shampoo 4% and normal shampoo.

a) b)

c) d) Figure 4. Scanning electron photomicrographs of virgin Asian hair: a) without any treatment; b) exposed to dust without any pre-treatment; c) exposed to dust after normal shampoo washing for 2 times; d) exposed to dust after active shampoo 4% washing for 2 times.

6. MO seed extract in hair conditioner. After confirmed that MO seed extract in shampoo system at moderate dosage as 2% can help the bleached hair repel the dust deposition, it is then put into the conditioner system toward a same objective. It was introduced into conditioner as 1% with the assumption of the better active delivery efficacy in this vehicle. 200g/m3 of dust exposure was applied in 3 minutes to the bleached hair tresses, and quantified of their gloss properties by the luster parameters. In the meantime, ICP analysis was conducted to quantify the real amount of dust deposited. Our hypothesis was then proved right, as shown in Figure 5 and Table 11 that 1% MO seed extract is sufficient to demonstrate an improvement on hair gloss from a vehicle of hair conditioner. The normal conditioner though increased the gloss before dust exposure, from both the luster parameters

Nc and LReich-Robbins. It did not show the benefit to repel the dust, according to the lowest LReich-Robbins after dust exposure . Nevertheless, the dust amount as presented by SiO2% from ICP analysis is ~0.02% lower if compared the tresses treated by the active conditioner with normal conditioner(Figure 6). A separate panel evaluation also indicates the effect of active conditioner on increasing the gloss/combility. Both the conditioners resulted in a higher dust deposition on hair compared with the non-treated one from the ICP result, which is interesting showing the same trend suggested by a series of shampoo tests. And further ICP analysis is to be carried out, to verify the possible higher dust deposition from shampoo washing than no-treatment.

a) b) c)

d) e) f)

Figure 5. The specular light image of the hair tress before/after(left/right) dust exposure: a) treated by nothing; b) treated by normal conditioner; c) treated by active conditioner 1%; The diffuse light image of the hair tress after dust exposure: d) treated by nothing; e) treated by normal conditioner; f) treated by active conditioner 1%.

Table 11. The luster parameters Nc and LReich-Robbins for the hair tresses treated by conditioners, before and after dust exposure.

No treatment Normal conditioner Active conditioner 1% Parameters Before After Before After Before After

Nc 0.6348 0.598255 0.673451 0.565117 0.664586 0.631947

LReich-Robbins 0.102354 0.055826 0.102054 0.045362 0.050735 0.054761

Figure 6. The SiO2 w.t.% as to the hair tress weight analyzed by ICP method.

Conclusion We have utilized various approaches to qualify and quantify the dust deposition on bleached/virgin Asian hair. Based upon the equipment measurement results, it was deduced that normal shampoo with the composition in this study may not be helpful to repel the dust deposition for bleached Asian hair. Panel evaluation gave the same conclusion. Regarding the effect of MO seed extract, it is proved by all the methods in this study, as an effective technology to be applied in shampoo format towards the protection of bleached Asian hair. The improvement on gloss and combility on bleached Asian hair can be ascribed to the higher deposition of MO seed extract due to its cationic nature, while on virgin hair only a negative effect of this extract on combility was noted with significant difference. At the same time, the SEM micrograph did not show an obvious reduction of dust deposition on virgin hair by using the active shampoo. We further tried this extract in the conditioner base, and used ICP method to quantify the dust amount deposited. The result correlated well with what we saw from the shampoo system, meaning the active effect on the bleached Asian hair is robust and will have great potential to apply to versatile vehicles.

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