The Effect and Mechanism of Transdermal Penetration Enhancement of Fu’s Cupping Therapy: A New Physical Penetration Technology for Transdermal Administration with TCM Characteristics

Background: in this paper, a new physical penetration technology for transdermal administration with traditional Chinese medicine (TCM) characteristics Fu’s cupping therapy (FCT) was established and studied by in-vitro and in-vivo experiments; the penetration effect and mechanism of FCT physical penetration technology (FCT-PPT) was preliminarily discussed. Method: Indomethacin (IM) as a model drug,by transdermal in vitro tests the establishment of the high,medium and low reference were finished as the chemical permeation system; chemical penetration enhancers and iontophoresis as a reference , the percutaneous penetration effect of FCT for IM patch was evaluated with 7 species diffusion kinetics model and in vitro drug distribution;naproxen as an internal standard,using UPLC-MS/MS technology , the IM quantitative analysis method in vivo was established , and pharmacokinetic parameters (AUC0-t,AUC0-∞,AUMC0-t,AUMC0-∞, Cmax and MRT) as indicators were used evaluate to FCT penetration role in vivo;in the same time,the group used 3K factorial design to study joint synergistic penetration effect on FCT and chemical penetration enhancers (CPEs);by SEM and TEM,the skin micro and ultrastructural changes of the stratum corneum (SC) surface were observed, to explore pay tank penetration mechanism. Results: In vitro and in-vivo skin permeation experiments revealed that both the total cumulative percutaneous amount and in-vivo percutaneous absorption amount (AUC and AUMC) of indomethacin that permeated SD mouse skin using FCT techniques were greater than the amount observed using CPE and iontophoresis: Firstly, in contrast to the control group, the indomethacin percutaneous rate (PR) of the FCT lower group (FCTL) was 35.52%, and the enhancement ratio (ER) at 9h was 1.76X, which was roughly equivalent to the penetration enhancing effect of the CPEs and iontophoresis; secondly, the indomethacin PR of the FCT middle (FCTM) group and the FCT high intensity group (FCTH) were Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 15 December 2016 doi:10.20944/preprints201612.0082.v1

Example of FCT treatment on human skin Fig.2 Fu's cupping made of white rubber development of TCM cupping therapy, encloses and creates a confined space between the body skin (or mucosa) and the walls of cup through FCT effect, namely forming a confined space between the body skin (or mucosa) within the FCT treating skin area and the walls of cup, which shapes and serves as an artificial environment of FCT producing various stimulating effects on human skin and subcutaneous structure. And multiple physical, chemical and biological effect parameters within the cup cavity, such as pressure, temperature, and amounts of O2, CO2, can be observed and regulated through its observation slot and regulation slot on the cup respectively. Since the cup cavity with small size is constructed artificially that presses on the body, it is called and referred to as "the artificial near-body microenvironment full regulation therapy", namely the theory of "Generalized Meridian -Three Fields and Two Cavities-Gap", which all are part of TCM theories. And FCT's creating several effects on human body as a TCM treatment, especially Skin, have been revealed via modern researches. Among them, several aspects may account for support and interpret this idea of FCT being used as a method of drug enhanced skin penetration.
Based on the above, we focused on the preliminary exploration of utilizing FCT and creating a new physical technology for transdermal administration with TCM characteristics. In our work, 1-(4-chlorobenzoyl)-5-methoxy-2-methylindol-3-ylacetic acid (indomethacin, a model drug) hydrophilic gel patch [13,14] was prepared as the preparations for transdermal administration.
Firstly, compared with CPEs (including 3%Azone and 5%Azone) and iontophoresis, the penetration enhancement effects of FCT (divided into three groups: FCTL, FCTM and FCTH) on indomethacin hydrophilic gel patches were evaluated through the in-vitro and in-vivo transdermal penetration enhancement tests，in which in-vitro transdermal parameters and in-vivo pharmacokinetic parameters were used as the detection indexes. Posteriorly, the synergism between FCT physical penetration technology and chemical penetration enhancers, concerned about those various ratios of CPEs, in transdermal enhancement were investigated initially, utilizing the 3 K factorial design and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technologies. Furthermore, Skin treated with chemical penetration enhancers, iontophoresis and the FCT, obtained from in vitro skin penetration studies, was examined via using scanning electron microscope (SEM) [15,16] and transmission electron microscopy (TEM) [5,9,[15][16][17][18][19][20] to investigate the effect of FCT-PPTs on ultrastructural changes of the skin, especially its SC, and to evaluate tentatively and preliminarily the mechanism by which FCT enhances skin penetration.

Materials
Indomethacin and naproxen references (purity > 99.9%, determined by HPLC) were obtained from National Institutes for Food and Drug Control. HPLC grade Acetonitrile, methanol and formic acid were all obtained from Tedia Company Incorporation (Fairfield, USA). Double distilled 4umulativ water was prepared through a Milli-Q water purification system (Millipore, USA). Indomethacin hydrophilic gel patches were prepared and supplied by Preparation Laboratory of Guiyang College of Traditional Chinese Medicine. Hydrophilic gel matrix materials, used to prepare indomethacin patches, were purchased from Shanghai Ye source Biotechnology Co., Ltd. Sprague-Dawley (SD) rates of SPF grade, weighing 320 to 350 grams (g), were supplied by the Experimental Animal Center of Third Military Medical University (Chongqing, China).

Preparation of indomethacin patches
By screening and optimization ， prescription of indomethacin matrix were identified; its composition was PVA of 17.350g, gelatin of 9.999g, PVP of 6.260g, CMC-Na of 8.140g, carbomer-940 of 3.000g, glycerol of 12.880g, propylene glycol of 12.880g, triethanolamine of 4.200g.
And the preparation process of matrix patches was as follows: firstly, PVA and gelatin weighed in prescription were properly swollen in the water overnight, heated by a water bath heater and then prepared as the mixed gel solution A (MGSA); secondly, PVPK-30, CMC-Na and carbomer-940 were weighed, mixed, swollen, dissolved, heated and prepared as the mixed gel solution B (MGSB); thirdly, after the weighing and mixing of glycerol, propylene glycol, triethanolamine, model drugs were added and stirred until these drugs were completely dissolved as the mixed gel solution C (MGSB); fourthly, the MGSB and MGSC were added in the solution A correspondingly, stirred while being added, and the water was added until the all solution weighs 600g, evenly stirred, and deposited to remove the air bubbles; in the end, the final MGS was quantitatively poured into a rubber ring attaching to blank matric backings (as long as 7 cm in diameter), extended and frozen for 2h, dried in natural conditions, until a total of 30 patches were formed with the same uniform, thickness and unified colors, and the dosage per patch was 12.5mg. cup, the speed should not be too fast, shake at an appropriate angle, and operate once every 2s.
Moving cupping, also known as slipping or scraping cupping, often applied the larger part of lesions or muscular part, namely apply a layer of massage oil or glycerin on such part, press the cup on skin with the left hand, make it tightly secured, pull the cup with the right hand at one direction to make it slip along the meridians, until the skin flushes.
Based on this procedure, experiments of FCT penetration enhancement were divided into three groups: the high, middle and low intensity group of FCT penetration enhancement，abbreviated as FCTL，FCTM and FCTH，were formed as shown in Table 1.

In-vitro transdermal test
The Franz in-vitro transdermal diffusion device [15,20] was used to carry out in vitro transdermal experiments [2] with the volume of receiving pool (V, 7.0ml) and the contact area (A, 2.92cm 2 ), divided into 8 groups, i.e.: the blank group, the 3%Azone group, the 5%Azone group, the 3%Azone-5%Mint oil group (dualistic enhancers, namely DCPEs group), the iontophoresis group, the FCT groups, processed as per the requirements of each group. And patches were used to administer drugs.
First of all, 20% ethanol PBS solution were used as receiver solution, and the skins of SD rats were used as percutaneous medium; after that, the skin was tightly fit to the patches and fixed 2 1 3 4

5
on the diffusion pool. Secondly, the receiver solution and its watery environment were completely adjusted to the constant temperature at 37℃±1℃, electromagnetically stirred at the constant speed of 320r/min; at last, we immediately filled up the control receiver solution after sampling and fully withdrawing at 3h, 6h, 9h, 12h, 16 h, 24h, 30h and 36h respectively. After such treatment, the sample were determined analyze the drug concentration (C, μg/ml) at each time point, and total cumulative penetration amount at all points and the cumulative penetration amount per unit area (QA, μg/cm 2 ) were calculated as shown in Equation 1.

Index detection
According to the in-vitro cumulative transdermal penetration amount [18] of indomethacin (Q, μg) at each point; the ratio of cumulative transdermal penetration amount to total dosage of indomethacin was calculated, namely the cumulative penetration percentage (Q%); and the  Table 2.
After the completion of experiments, the matrix patch and skin were removed and cut into pieces, and then extracted for 90min using 50ml methanol and heating reflux, extracted once; and then the indomethacin residual in the matrix patch and in skin with each extraction solution was detected to obtain the indomethacin distribution in paste, skin and receiver solution was shown as Fig.7.

Quantitative method
The analysis was performed on a WATERS Xevo TQ UPLC-MS/MS system (Waters, USA), where the chromatographic column of ACQUITY UPLC Shield RP (C18, 1.7μm, 2.1×50mm, Waters, USA) and the mobile phase of the acetonitrile-0.05% formic acid was used to enables complete separation of target components separate under the gradient elution (Table 3). Simultaneously, the total ion chromatograms(TIC) and quantitative data were recorded and processed by using an electrospray ion source [21] under the mass spectrum conditions: the ion source temperature  Table   4 for the ion peak spectrum conditions.   In-vivo percutaneous penetration enhancement test SD rates were chosen and randomly divided into 5 groups, i.e.: the control group, the FCTM group, the FCTL group, the CPE-3%azone group and the CPE-5%azone group. After the intraperitoneal injection and anesthesia using 10% urethane, rats were unhaired and the shu-back points were marked and treated as per the requirement of each group; in addition, the work area 3.14cm 2 were selected within the marked part of skin, and used to administer drugs with the patches dosage of 2.0mg/rat; the 3%azone patches were delivered to the chemical enhancer group, the none-enhancer patches were used to the control group, and the patches were used to administer drugs after cupping therapy treating in the FCT group.
The capillary (the sodium treatment was done using heparin) with diameter of 1.0mm was used to take 0.5ml blood from the venous plexus under the eye sockets of rats; the whole blood samples were taken at 1h, 3h, 6h, 9h, 12h, 16h, 20h, 24h, 28h, 36h and 48h respectively, then placed into anticoagulation EP. And plasma was prepared by centrifugation. 100 μl of rat plasma were mixed with 700 μg of internal standard, and 1ml of ethyl acetate was added into it to extract. The mixture was vortexed for 10 minutes and then centrifuged at 12,000 rpm for 13min in a centrifuge at 10℃. Then 800 μl of supernatant raffinate was be transferred to into a nitrogen blowing concentrator (Tianjin Automatic Science Instrument Co., Ltd, Tianjin, China) to evaporate, until turned into a residue. The residue was dissolved with 0.5ml of acetonitrile, then used to detect via the UPLC-MS/MS system.

Concentration-time curve of each group
According to the blood concentration (C, ng/l) of each group, the concentration (C, ng/l)-time (t, h) curve of each group was drawn. And in contrast, the findings showed that the C-t curve of FCTM group was significantly higher than that of any other group, while the concentration-time curve of FCTL group was slightly higher than that of the chemical penetration enhancer group ( Fig.8).

Penetration enhancement synergism study
The synergism of FCT penetration enhancement and CPE was examined and using a 3 K factorial design: the chemical enhancer was used as "  Table 5. glutaraldehyde was used to solidify, and placed in a refrigerator to preserve and solidify at 2～4℃ for 24h. The 2mm×2mm tissue block was dehydrated, dried and "metal spraying" plating, then the surface structure of skin SC was observed by a JSM-6940 SEM (Hitachi, Japan); after the 1mm×1mm tissue block was rinsed, dehydrated, soaked, embedded and treated in other methods using 0.1mol/LPBS, the ultra-thin sections were prepared and stained, and the SC ultrastructure were observed via a Hitachi-7650 TEM (Hitachi, Japan).

Analysis of in-vitro results
The in-vitro results ( Fig.10 and Fig.11) showed that FCT had a significant effect on the transdermal penetration enhancement: Firstly, compared with the control group, the indomethacin percutaneous ratio (Fig.12)

Analysis of in-vivo pharmacokinetic results
According to the concentration-time curves (Fig.13) (Table 6 and Table 7). The findings showed that AUC and AUMC of in-vivo percutaneous absorption as a result of FCT penetration enhancement were higher than that of the control group; AUC and AUMC of the FCTL group were slightly higher than that of the CPE group; the AUC and AUMC of the FCTM group were significantly higher than that of any other group, which indicated that FCT had a facilitating effect on IM transdermal absorption. interaction A×B (P, Table 8) was beneath 0.0001 and its difference was markedly significant, indicating that A×B interaction has a markedly significant effect on the in-vivo percutaneous absorption of IM; if combined with the A×B interaction visual chart (Fig.10), it demonstrated that the combination of the FCT and CPEs generated a significant synergistic effect (synergism) on the penetration enhancement of indomethacin. And the independent analysis findings of each index were consistent with the analysis results of normalization values.

Analysis of SEM images
The SEM images were as shown in Fig.11 (A-F). The figures were ×200X with 400×300 pixels, which were reduced to 60.0×70.0mm.The control group images indicated that the SC surface cells of SD rats were flat, relatively regular, and closely spaced with some ridgelines and zigzagging arrangement, as shown in the yellow-arrow part (Fig.11 A).
In comparison with the control group ( Fig.11 A), the FCTL (Fig.11 B) and the FCTM (Fig.11 C) images had more cracks, loose and irregular arrangement on the SC surface structure; meanwhile, the SC surface presented the microstructure slices and the skin SC pores grew bigger as shown in the red-circled part; and with the increase of FCT penetration enhancement, the SC structure fissure grew partly bigger as marked in the yellow-arrow part.
The SC surface of SD rats in the FCTH (Fig.11 D) was almost completely damaged, hard to observe the complete SC surface structure. These revealed that dense structure of the SC has been damaged to varying degrees by diverse FCTs, which was probably part of the percutaneous penetration mechanism.
Compared with the control group ( Fig.11 A), the CPE-3%azone group (Fig. 11E) and the CPE-5%azone group (Fig.11 F) had slightly bumpy wrinkles, relatively loose arrangement and irregular arrangement on the flat surface structure of the SC; with the increase of CPE penetration enhancement, the SC grew bigger as marked in the yellow-arrow part.
By contrast with the FCT groups, the SC surface of SD rats treated by CPEs had internal bumpy wrinkles that were clearly shown in Fig.11 E and Fig.16 F, and had no significant cracks with relatively loose arrangement, but less clearly shown than the FCT group; besides, the SC surface pores didn't grow bigger evidently as marked in the red-circled part.
Furthermore, FCT high intensity group damaged SC and the lower portion of vascular structure (vessels and microangiums) beneath SC was observed as marked in the redarrowed part (Fig.12), depriving or decreasing its barrier function, and consequently the in-vitro animal skin had cracks after 24h during the in-vitro percutaneous absorption experiments, which made it impossible to go on further percutaneous experiments.

Analysis of TEM observation images
The control group, FCT low intensity group, FCT middle intensity group, chemical enhancer low intensity group, and chemical enhancer middle intensity group were shown in Fig.13 (A-D) respectively; the 16×3,000X figure was obtained from Hitachi TEM System, the size of which was reduced to 70.0×67.8mm.The control group (Fig.13A) indicates that the keratinocytes SC ultrastructure of normal SD rats had the compact "brick structure", which was regularly and closely arranged, overlaid layer by layer, with lipid composition between the layers that was densely spaced and flat-shaped; it constitutes the complete lipid film with compact structure, low water content and high resistance, which has the function of barrier protection and is considered to be the major obstacle to the transdermal absorption of drugs as indicated in the yellow-arrow position.
Compared with the control group, after the treatment of FCT penetration enhancement as shown in Fig.13B, the "brick structure" from SC became loose, the gap increased, with the irregular arrangement and the lower degree of overlaying, and such changes can reduce the resistance of drugs penetration and increase the transdermal absorption of drugs.
Compared with the control group, the SC ultrastructure after the treatment of chemical penetration enhancer ( Fig.13C and Fig.13D) had the loose arrangement, the gap was reduced, but this change was less clear than FCT group, and the intersecting degree of keratinized structure insignificantly reduced as indicated in the yellow-arrowed position.

Discussion
This research attested that as a new physical technology for transdermal administration, FCT can enhance the IM percutaneous absorption through the in-vitro and in-vivo percutaneous experiments; meanwhile, the FCT and CPEs had a markedly synergistic effect on penetration enhancement. SEM and TEM results showed that the SC structure significantly changed after the treatment of penetration enhancement, which reduces the barrier function and increases the percutaneous absorption of drugs; thus it has been verified that the first action mechanism of FCT-PPT is closely interrelated to its physical effects (negative pressure), and it did not involve other action mechanism of percutaneous penetration enhancer.
Firstly, its physical effects, resulting from subatmospheric pressure of FCT, make the fissures, keratinocyte proliferation and acidic liquid secretion evenly scattered on skin epidermis through stimulating effects of heat, force, bioelectricity and other physical factors; secondly, its chemical effects, stemming from changes of chemical substance body and fluids in the body, treat various acute and chronic diseases and play a role in the prevention of diseases and health care by the combined therapy of drug therapy, Chinese herbal medicine and cupping therapy; thirdly, biological effects and other stimulating effects, including bioelectric effect, opening and closing of ion channels (such as Ca 2+ ), have a comprehensive regulation on the whole human body and SC barrier functions through the changes of physical and chemical properties and conditions, neural activities and body fluid metabolism due to the meridian stimulation, angiectasis, blood flow increase, enhanced permeability of partial biofilm system and other effects, resulting in the overall responses as shown in 14~17.

Conclusion
Therefore, it is considered that FCT as a new physical technology for transdermal administration with TCM characteristics has a significant effect of penetration enhancement as well as a high value of TCM clinical application, which is worthy of being disseminated and developed; its potential mechanism of penetration enhancement and FCT stimulation are closely associated with the change of SC and the biological regulation of skin. But its functional mechanism of penetration enhancement needs to be further researched by fluorescence micro-imaging techniques and various TCM model drugs are required to further validate the effects and features of FCT penetration enhancement.