C. militaris extracts
The external appearance of each C. militaris extracts were different. CC, CA, and CE were brown-yellowish semisolid masses with a distinct odor on the outside, while CH was a lighter brown-yellowish semisolid mass. In contrast, CW was a light-brown dry powder.
Anti-wrinkle activities of C. militaris extracts
Skin ageing is naturally occurred by a decline of the extracellular matrix (ECM), including collagen fibers, elastin fibers, and hyaluronan by the favor of MMP-1, elastase, and hyaluronidase, respectively [18]. These enzymes mostly presented in the dermis layer and played important roles in the degradation of ECM and resulting in the skin winkles [19]. Inhibitory activities against MMP-1, elastase, and hyaluronidase of C. militaris extracts are shown in Table 2. Among different C. militaris extracts, CW possessed the significantly highest MMP-1 and elastase inhibitory activities with the inhibition of 77.9 ± 5.3% and 84.4 ± 4.0%, respectively. Interestingly, the MMP-1 and elastase inhibition of CW was as potent as oleanolic acid and EGCG, a naturally occurring triterpenoid widely known for MMP-1 and elastase cascade [11]. Therefore, CW was suggested as a potent natural extract which exerted anti-wrinkle activities. On the other hand, cordycepin was noted as a major component of CW which responsible for MMP-1 and elastase inhibition since it exhibited potent MMP-1 and elastase inhibitory activities with the inhibition of 58.5 ± 4.9% and 76.5 ± 7.1%, respectively. Although, there are some previous reports revealed about the MMP-1 inhibitory activity of C. militaris, they suggested cordycepin as the key compound that inhibited the activator protein-1 and resulting in the suppression of MMP-1 expression [20, 21]. However, the present study was the first to remarked that both cordycepin and C. militaris extracts exerted a direct effect on the MMP-1 enzyme. Additionally, the present study was the first to reveal anti-elastase of C. militaris extracts and suggested CW as the most potent anti-wrinkle ingredient for cosmetic formulations. However, the IC50 value was preferable to comparing the strength of the inhibition of CW with the standard compound. Therefore, it was suggested for further study to investigate the IC50 value of CW in a comparison with oleanolic acid and EGCG on the inhibition of collagenase and elastase, respectively.
Table 2
Anti-ageing activities of C. militaris extracts
Samples | Collagenase inhibition (%) | Elastase inhibition (%) | Hyaluronidase inhibition (%) |
OA | 71.7 ± 0.2a | N.D. | N.D. |
EGCG | N.D. | 89.6 ± 4.1a | 74.8 ± 2.3a |
COR | 58.5 ± 4.9b | 76.5 ± 7.1a,b | 65.5 ± 2.1a |
CH | 0.0 ± 0.0c | 32.9 ± 0.2d | 30.7 ± 8.5b |
CA | 0.0 ± 0.0c | 55.8 ± 8.5b,c | 69.0 ± 3.9a |
CE | 56.6 ± 3.0b | 42.4 ± 5.0c,d | 33.2 ± 7.6b |
CC | 10.8 ± 0.9c | 26.5 ± 7.3d | 41.5 ± 7.1b |
CW | 77.9 ± 5.3a | 84.4 ± 4.0a | 14.3 ± 5.6c |
Note: OA = oleanolic acid; EGCG = epigallocatechin gallate; COR = cordycepin; CH = hexane extract; CA = ethyl acetate extract; CE = ethanolic extract; CC = crude ethanolic extract; CW = water extract; N.D. = not determined. The final concentration of all tested compounds and extracts in the tested system was 0.1 mg/ml. Data are shown in mean value ± S.D. (n = 3). There letters a, b, c, and d denoted significant difference between samples analyzed using post-hock Tukey ANOVA (p < 0.05). |
In contrast to MMP-1 and elastase inhibition, CW possessed the least inhibitory activity against hyaluronidase. CA was noted as the most potent hyaluronidase inhibitor with the inhibition of 69.0 ± 3.9%. Interestingly, CA was as potent as EGCG which was well-known as anti-ageing compound. Although cordycepin possessed a potent inhibitory activity of 65.5 ± 2.1% against hyaluronidase, the cordycepin content was not related to the hyaluronidase inhibition. Therefore, cordycepin was not the only compound responsible for the hyaluronidase inhibitory activity. Previous studies suggested that C. militaris contained large quantity of polysaccharides [22, 23], which has been reported for the hyaluronidase inhibition [24]. Since the retardation in hyaluronan degradation and increasing hyaluronan level in the skin layer by both putting hyaluronan in topical skin care products or using as a temporary dermal filling agent were suggested for plumping and youthful skin [25]. CA would be another natural extract potentially retard the skin ageing.
Although CA possessed the significantly highest hyaluronidase inhibition (p < 0.05), it had no effect on MMP-1 inhibition. Therefore, CW was selected for further formulations development since CW exerted the most potent MMP-1 and elastase inhibition (p < 0.05), as well as a moderate hyaluronidase inhibitory activity (14.3 ± 5.6%). The effective concentration of CW was suggested at 0.1 mg/ml because this concentration led CW to possess approximately 80% inhibitory activity against both MMP-1 and elastase. However, only some active compounds could release and deeply penetrate into the target site of the skin. Therefore, approximately 100 times higher than the effective concentration, which was 10 mg/ml or 1% w/v, was suggested for further product development [6].
Chemical characteristic of CW
CW, which was the most potent anti-wrinkle extract, was characterized for its chemical constituent. The HPLC chromatograms of cordycepin and CW are shown in Fig. 1. Cordycepin was detected at the retention time of 6.347 min, whereas, there was a major peak detected around 6.3 min in the HPLC chromatograms of CW. Therefore, it could be noted that cordycepin was a major chemical constituent of CW, which could be further used as a marker for the quantitative determination of CW in the further study.
Microemulsions development
Effect of oil type
Various oil types generated different microemulsion region in the pseudoternary phase diagrams as shown in Fig. S1. Sugar squalane gave the largest area of microemulsion (9.5%), followed by jojoba oil, argan oil, apricot kernel oil, canola oil, corn oil, avocado oil, and perilla oil, respectively. Since sugar squalane is a unique cosmetic ingredient which is a mobile, colorless, odorless, and has good physical and chemical stability, it was selected for the further studies. Additionally, squalene has been known as a natural triterpene hydrocarbon which is one of the most important lipids in human skin cell and accounted for up to 13% of total lipids synthesized from the sebaceous glands [26].
Effect of surfactant type
Pseudoternary phase diagrams constructed using various types of surfactant. Tween® 85 was the only one surfactant which could generate microemulsion with the region of 9.5% in the pseudoternary phase diagram. The results were well accordance with previous studies, which suggested Tween® 85 as the most suitable surfactant for microemulsion development [27, 28]. Therefore, Tween® 85 was selected for the further studies.
Effect of co-surfactant type
The single surfactant is not enough for reducing interfacial tension between water and oil and generate microemulsion, of which the internal droplet size is in the range of 10–200 nm [29]. Therefore, co-surfactant is required in the development of microemulsion. Pseudoternary phase diagrams constructed using various types of co-surfactant are shown in Fig. S2. The results showed that propylene glycol gave the largest microemulsion area in pseudoternary phase diagram (11.1%), followed by butylene glycol. On the other hand, glycerin could not generate microemulsion. The likely explanation might be due to the presence of alkane triol which composed of three hydroxyl groups in the glycerin molecule and led to high hydrophilic property but lower efficacy in the interfacial tension reduction [30]. Therefore, propylene glycol was selected for the further studies.
Effect of surfactant to co-surfactant ratio
Various ratio of surfactant to co-surfactant showed different microemulsion region in the pseudoternary phase diagrams as shown in Fig. S3. The results represented that microemulsion region increased when the surfactant proportion in Smix increased from 1:2 to 2:1. Therefore, it could be concluded that there should be higher content of surfactant than co-surfactant in the Smix. However, microemulsion region tended to decrease after increasing of the surfactant from 1:2 to 5:1. The likely explanation might be due to the lower co-surfactant content which was not enough to reduce the interfacial tension. Therefore, Smix ratio of 2:1 was selected for further studies.
Microemulsion preparation
Since all parameters, including oil type, surfactant type, co-surfactant type, and Smix ratio affected the microemulsion region in the pseudoternary phase diagrams, the system that generated the largest region of microemulsion was selected for further microemulsion development. In brief, the selected system composed of sugar sqaulane, Tween® 85, propylene glycol, and DI water. The Smix ratio was 2:1. Four formulations (ME1 – ME4) along the line of 30% w/w water content as shown in Fig. 2 were developed and characterized to investigate the effects of different Smix and oil content.
All formulations (ME1 – ME4) were homogeneous transparent liquid with low viscosity which could be defined as microemulsion [31]. Higher content of Smix led to darker yellow color and more viscosity. The internal droplet size of each microemulsions were ranging from 107.2 ± 2.9 nm to 366.3 ± 16.0 nm as shown in Fig. 3. Since all microemulsion contained 30% w/w of water phase, the increasing of Smix but decreasing of oil phase were used in ME1 to ME4, respectively. ME1, which contained the highest oil content (40% w/w), tended to be W/O microemulsion since the amount of oil phase was higher than the water phase. ME2, which contained an equal amount of oil and water phase (30% w/w), tended to be O/W microemulsion since Tween® 85 is hydrophilic surfactant with high HLB value (11.0). The results noted that ME2 had the smallest internal droplet size (107.2 ± 2.9 nm). However, the size of O/W microemulsion increased with the increasing amount of Smix although the oil content decreased. The likely explanation might be due to their higher viscosity since size of internal droplets has been reported to increase with an increase of the viscosity [32]. PDI of these microemulsions were ranging from 0.7 ± 0.1 to 1.0 ± 0.0, which were very high. The likely explanation might be due to the measurement of undiluted sample of which the internal droplets were not well dispersed and led to the detection of larger size than usual [33]. However, microemulsion could not be diluted before the size and PDI measurement because the microemulsion system would be changed. On the other hand, all microemulsion had the same pH value around 6.9 ± 0.1 to 7.1 ± 0.2. Additionally, all microemulsions were stable since no changing in external appearance, e.g., separation, sedimentation, or coagulation, were not detected after 8 cycles of heating-cooling condition. The internal droplet size was still remained the same. Therefore, ME1 was selected as a representative of W/O microemulsion and ME2 was selected as a representative of O/W microemulsion for further incorporation of C. militaris extract (CW).
Microemulsion containing C. militaris extract
ME1 and ME2 were selected for the incorporation of CW. The external appearance of microemulsion containing C. militaris extract (ME1-CW and ME2-CW) were homogeneously transparent and brown-yellow color with low viscosity. The internal droplet size of ME2-CW (146.1 ± 1.5 nm) was significantly smaller than that of ME1-CW (212.4 ± 1.5 nm) (p < 0.05). On the other hand, the PDI of ME2-CW (0.5 ± 0.0) was significantly narrower than that of ME1-CW (0.9 ± 0.1) (p < 0.05). Therefore, ME2-CW tended to be more stable than ME1-CW due to its narrower size distribution of the internal droplets. Interestingly, the internal droplet size of ME1-CW and ME2-CW were larger than their own blank microemulsions as shown Fig. 4. Hence, it could be assumed that CW was entrapped inside the droplet of microemulsion and led to larger internal droplet size after the C. militaris extract incorporation.
A basic analysis of the stability under heating-cooling conditions could be used as a fast tool to predict the stability of emulsions, such as cosmetic formulations [34, 35]. After 8 cycles of heating-cooling condition, both ME1-CW and ME2-CW were physically stable since no changing in external appearance were detected. However, the internal droplet size of ME1-CW was significantly increased (p < 0.05). The results were well accordance with the previous PDI data noted that the internal droplet size distribution of ME1-CW was board. On the other hand, ME2-CW was stable since its internal droplet size was still remained unchanged. Although the internal droplet size of ME1-CW increased, the size was still in the nano scale (212.4 ± 1.5 nm). Therefore, ME2-CW were selected for further preparation of topical serum formulation.
Cytotoxicity effects on HaCaT cells of microemulsion containing C. militaris extract
The cytotoxicity effects on HaCaT cells of microemulsions with and without C. militaris extract (CW) are shown in Fig. 5. All formulations were found to be safe since the HaCaT cell viability was approximately higher than 80%. No significant difference was detected among these formulations. The HaCaT cells viable after being exposed to the aqueous solution of C. militaris water extract (CW), ME1, ME2, ME1-CW, and ME2-CW were 78.5 ± 1.3%, 86.26 ± 1.0%, 81.6 ± 0.0%, 80.2 ± 1.9%, and 79.4 ± 4.6%, respectively. The results were well comparable with our previous study which reported that nanoemulsions had no cytotoxicity effect on the HaCat cells and biocompatible [6]. Therefore, both ME1-CW and ME2-CW could be used for the preparation of topical serum formulations for evaluation in human volunteers. ME2-CW, on the other hand, with a smaller internal droplet scale and a narrower PDI, was chosen for further production of topical serum formulations.
Topical serum formulation from microemulsion containing C. militaris extract
The external appearance of topical serum formulation from microemulsion containing C. militaris extract was homogeneously transparent yellow liquid. The viscosity was 0.8 ± 0.0 mPas. The formulation was stable since no changing in external appearance, e.g., separation, sedimentation, or coagulation, were not detected after 8 cycles of heating-cooling condition. Additionally, the viscosity was still remained the same. Therefore, it was a good formulation suggested for further applied in the human volunteers.
Microbial enumeration test of non-sterile serum formulation
Microbial contaminants may grow by using ingredients in the products causing physicochemical changes or spoilage and finally loss in product safety, quality, activity, and stability. In tested serum, the TAMC was ≤ 1 x 102 CFU/mL and the TYMC was not detected. The specified pathogens, E. coli, P. aeruginosa, S. aureus, Clostridium sp. and C. albicans were not detected in tested serum formulation. The tropical serum products were compliant with the accepted criteria according to guidelines of non-sterile and cosmetic products due to non-excessive microbial count and the absences of specified pathogens. The compliant serum formulation was applied use in human volunteers.
Irritation properties of topical formulation from microemulsion containing C. militaris extract
Thirty healthy human volunteers (77% of female and 23% of male), ranged in age from 21 to 60, were tested on the serum formulation. The results noted that the topical formulation from microemulsion containing C. militaris extract induced no irritation sign on human skin. Therefore, it was suggested as safe for using topically on the skin.
Efficacy of topical formulation from microemulsion containing C. militaris extract
After two weeks of the application, the skin appearance improved as shown in Fig. 6. The results showed that the skin elasticity and skin moisture significantly improved after 1 week of serum products application and continuously increased after 2 weeks as shown Fig. 7. Interestingly, the topical formulation from microemulsion containing C. militaris extract could enhance the skin elasticity and skin moisture when compared to its own base formulations. The topical formulation from microemulsion containing C. militaris extract could improve the skin elasticity by 39.9 ± 7.3% from the baseline after 1 week and maintained at 38.5 ± 9.0% after 2 weeks of application. The potent inhibitory activities of CW on MMP-1 and elastase may be the likely explanation of the effective results on the skin since it could reduce the damage of collagen and elastin fiber. Apart from the enhancement of skin elasticity, the skin moisture was also improved after the application of serum formulation. The topical formulation from microemulsion containing C. militaris extract could increase the skin moisture by 42.2 ± 14.2% from its baseline after 1 week and 38.3 ± 15.2% after 2 weeks of applications. The likely explanation might be due to the composition of various polysaccharide in the C. militaris extracts, which could reduce transdermal water loss (TEWL) and protect the skin barrier [36]. Although the skin moisturization measure in the present study was based on the electrical conductance, the reduction in TEWL would result in increased skin moisturization since water can be retained in the skin layer. Apart from C. militaris extracts, sugar squalane, which was used as an oil phase in the microemulsion, would be another component that would help to moisturize the skin. Sugar squalene is commonly used as an occlusive ingredient, so it could reduce TEWL and thereby in-creases the skin moisture. Therefore, the serum formulation not only effectively enhanced the skin moisture, but also improved the skin elasticity. Although the clinical studies regarding cosmetic formulations efficacy are usually performed for two or three months because the epidermal transit time is around one month, the skin improvement could be detected since the first week of a serum formulation application. The likely explanations were not only from the inhibitory activity on extracellular matrix degradation in the dermis layer but also attributed to the enhancement of the skin moisturization.
Satisfaction on the topical formulation from microemulsion containing C. militaris extract
The topical formulation from microemulsion containing C. militaris extract gained high level of the volunteers’ satisfaction after two weeks of application in the term of physical appearance and efficacy as shown in Fig. 8. It obtained a satisfactory score in terms of viscosity (4.4 ± 0.6/5.0), odor (4.5 ± 0.8/5.0), moisturizing effect (4.6 ± 0.7/5.0), emollient (4.7 ± 0.5/5.0), skin absorption (4.6 ± 0.6/5.0), skin texture (4.6 ± 0.7/5.0), skin slippery (4.6 ± 0.8/5.0), no skin irritation (4.9 ± 0.3/5.0), skin elasticity enhancement (4.3 ± 0.8/5.0), winkle reduction (4.2 ± 0.9/5.0), and non- sticky (4.3 ± 1.1/5.0).