Scientific Study on LA Muscle's Sculpt

LA Muscle's Sculpt has been a best seller across the world for close to a decade. This very natural product is the best CLA (Conjugated Linoleic Acid) product you can get your hands on. It is great for muscle maintenance and especially for fat loss.

LA Muscle has always stated that Sculpt also has powerful anti-oxidant capabilities. We decided to put this to the test and fund a scientific study on Sculpt's anti-oxidant capabilities.

The result was fantastic in that Sculpt showed some tremendous potential as a serious anti-oxidant, great for your health. For those who want it simple: LA Muscle's Sculpt is good for you! For the science-heads, read on:



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Sponsored summer project, carried out at Kingston University

Aim:
To investigate the antioxidant potency of L.A. Muscle 500-mg conjugated linoleic acid capsules using two in vitro antioxidant assays.

Background

The aim of this research was to examine the antioxidant potency of L.A. Muscle 500-mg capsules of 300 mg conjugated linoleic acids (CLA) and 0.025% vitamin E (?-tocopherol) using laboratory assays. Two different free-radical scavenging (antioxidant) assays were used, namely the ABTS assay and the DPPH assay. Both assays are based on the ability of compounds with antioxidant potency to donate an electron to 'quench' the free radical used in the particular system. For the ABTS assay, the 2,2?-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS-+) is formed in the laboratory 24 hours in advance of the reaction and for the DPPH assay, the stable free radical 2,2-Diphenyl-1-picrylhydrazyl (DPPH-) is dissolved in ethanol from powder form 30 minutes prior to use. For these studies, the ABTS-+ method published by Re et al (1) and the DPPH- method published by Kim et al (2) were replicated and validated in the laboratory before being used.

Both ABTS-+ and DPPH- have absorption spectra which can be measured using UV-Vis spectroscopy. ABTS-+ has absorption maxima at 645nm, 734nm and 815nm. DPPH- has an absorption maximum at 517nm. Once mixed with the free radical, compounds with antioxidant potency can donate an electron to the free radical species, thus quenching it to form the non-radical species instead. This results in a reduction in the absorbance of the radical as measured by spectroscopy. In these studies therefore, antioxidant potency is measured as a function of the reduction in the absorbance of ABTS-+ at 734nm and the absorbance of DPPH- at 517nm. When each system contains an excess of the free radical, antioxidant potency is concentration dependent. This relationship was demonstrated in these studies by measuring the effect of concentration on antioxidant potency (as measured by the reduction in free radical absorbance). The standard antioxidant trolox was used to validate both antioxidant assays before use. Trolox is a water-soluble vitamin E analogue routinely used as a standard in antioxidant assays. In addition, ?-tocopherol was also used to establish the antioxidant profile of the pure compound. An attempt was also made to measure the antioxidant capacity of pure conjugated linoleic acids. The antioxidant potency of L.A. muscle CLA/ ?-tocopherol capsules was measured in both the ABTS and the DPPH assays.

Materials and Methods

ABTS Free radical: 0.0077g/mL of 2,2?-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was weighed out, diluted in water, and vortexed for one minute. 0.00135g/mL of potassium persulfate was weighed out and diluted in water. This solution was vortexed and allowed to sit in a sonicating bath for five minutes. The ABTS and potassium persulfate solutions were then mixed together at a ratio of 1:1 giving a final ABTS concentration of 7mM and potassium persulfate concentration of 2.5mM. The ABTS-+ in this solution showed the greatest stability 16-24 hours after being mixed. Figure 1 shows the production of the free radical as a function of time. The ABTS-+ radical was used 16-24 hours post-production by diluting 100-fold in 100% HPLC-grade ethanol, giving absorbance readings of 0.75+/-0.03 at 734nm.

Figure 1. Absorption spectra of ABTS-+ as a function of time with the lowest spectra relating to the shortest time post-production.

DPPH Free radical: 2,2-Diphenyl-1-picrylhydrazyl (DPPH-) was prepared by diluting 0.00478g of stock DPPH in 120 mL 100% HPLC grade ethanol giving a final concentration of 101 µM. This solution was allowed to stand in a dark space for 30 minutes before being measured at 517nm and then used.

Trolox: (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) was made up as a standard at varying concentrations (0, 2.5, 5, 10, 15 µM final concentrations in the ABTS assay; 0, 10, 20, 40 µM final concentrations in the DPPH assay) in 100% HPLC grade ethanol.

?-Tocopherol: (±)-?-Tocopherol was made up at varying concentrations (0, 10, 15, 20 µM final concentrations in the ABTS assay; 0, 10, 20, 40 µM in the DPPH assay) in 100% HPLC grade ethanol.

CLA: Linoleic acid, conjugated was made up at 0, 10, 15, 20 µM final concentrations in the ABTS assay, in 100% HPLC grade ethanol.

L.A. Muscle CLA/ ?-tocopherol Capsules: The liquid was extracted from the capsules and tested at varying volumes directly in the ABTS and DPPH radical scavenging assays at volumes of 0, 20, 40, and 60 µl for both assays. The weight of these volumes was also measured in order to equate the aliquots to ?-tocopherol amounts as per label claim.

Results
ABTS Assay:

The reduction in the absorbance reading of ABTS-+ was measured at 734 nm at exactly 1 minute and 5 minutes after mixing with each antioxidant solution. The reduction in absorbance was expressed as a % inhibition of the original absorbance of ABTS-+. Figure 2 shows the percent inhibition of 5 different samples of 10 µM Trolox from time zero to five minutes. The figure demonstrates there is no further significant reduction in the inhibition of ABTS-+ after one minute of mixing.

Figure 2. Percent inhibition of the ABTS-+ radical by 10 µM Trolox.
The absorbance readings for the DPPH assay were taken at 517nm at exactly one minute after the addition of each antioxidant solution.

Trolox was used as a standard each day and repeatable results were obtained. Trolox was used to prove the linear relationship between antioxidant concentration and percent inhibition as seen in figure 3.

Figure 3: Trolox inhibition of ABTS-+ radical.

In addition, ?-Tocopherol and the L.A. Muscle capsules were then tested against the ABTS-+ radical. Both showed linear relationships in terms of inhibition versus concentration (figure 4).

Figure 4: Antioxidant potency of L.A. Muscle capsules and ?-Tocopherol expressed as ?-Tocopherol concentration (label claim for capsules).

The pure CLA purchased for the purpose of these studies proved to be very unstable. Pure CLA is sensitive to moisture, light and must be stored below -20oC and we were not able to obtain repeatable data for CLA within the ABTS assay. It appeared, by analyzing our results that the CLA degraded rapidly, most likely through oxidation. This fits in with the reported antioxidant potency of CLA - all antioxidants can oxidise. This finding also supports the designed formulation of the L.A. Muscle CLA capsules which contain the antioxidant ?-Tocopherol, encapsulated in a gelatine-like shell to protect against oxidation of the CLA within.

We do not therefore include any CLA results using the ABTS assay (because of unreliability) and could not test the antioxidant potency of CLA using the DPPH assay. Future studies of the antioxidant potency of CLA are planned - these will be carried out in a glove-box in anaerobic conditions using an inert gas instead of air (see 'Future work', below).

DPPH Assay:
Trolox was again used as a standard to prove the linear relationship between concentration and percent inhibition of the absorbance of the DPPH radical in this system (Figure 5).

?-Tocopherol and the capsule were then tested against the DPPH radical. Like the ABTS radical assay, both showed linear relationships in terms of inhibition versus concentration (figure 6).


Figure 5: Trolox inhibition of DPPH radical.



Figure 6: Antioxidant potency of capsules and ?-Tocopherol in terms of ?-Tocopherol concentration (label claim of ?-Tocopherol for capsules).

In both the ABTS and DPPH radical scavenging assays the ?-Tocopherol and L.A. Muscle capsules displayed reproducible results. The capsules had greater antioxidant potency than could be accounted for by the claimed ?-Tocopherol concentration within (at equivalent concentrations in comparison to antioxidant potency of pure ?-Tocopherol). This possibly means that the CLA within the capsules may be adding to the antioxidant potency of the vitamin E that is already present within. If the label claim of 0.025% vitamin E (?-tocopherol) is accurate, then there is an additional antioxidant within the L.A. Muscle capsules. CLA may have antioxidant potency as reported by Kim et al (2) and others but this remains to be quantified in our assays. The L.A. Muscle capsules also contain 200 mg of other polyunsaturated fatty acids - these may also have antioxidant potency although there is not much information relating to this in the literature.

What this means
In summary, if the label claim of 0.025% vitamin E (?-tocopherol) is accurate, the L.A. Muscle capsules show more antioxidant potency than can be accounted for by vitamin E alone in these capsules. This could be due to the presence of CLA within the capsules.

Weaknesses of the study
We did not measure the concentration of vitamin E within the L.A. Muscle capsules and have therefore not independently verified the label claim. We were not able to measure the antioxidant potency of pure CLA, because of the rapid degradation of the standard. We have not quantified the amount of CLA within the L.A. Muscle capsules.

Future work
This pilot study needs to be supported by quantification of vitamin E and CLA levels within the L.A. Muscle capsules. In separate studies, the antioxidant potency of pure CLA should be established. It would also be desirable to identify (if not quantify) the other PUFAs within the L.A. Muscle capsules.

Conclusion
The L.A. muscles CLA capsules do display antioxidant potency. The degree of the antioxidant potency is greater than comparable concentrations of pure vitamin E alone. It needs to be established whether this greater potency is due to higher concentrations of vitamin E than claimed on the label, or due to other components such as CLA within the capsules.

References
1. Roberta Re, Nicoletta Pellegrinie, Anna Proteggente, Ananth Pannala, Min Yang, and Catherine Rice-Evans. Antioxidant activity applying an improved ABTS radical cation decolorisation assay. Free Radical Biology and Medicine. 1999; 26 (9/10): 1231-1237.

2. Young Jun Kim, Ki Won Lee, and Hyong Joo Lee. Total antioxidant capacity of arginine-conjugated linoleic acid (CLA) complex. J. Agric. Food Chem. 2004; 52: 439-444